Journal: The Journal of Experimental Medicine

Article Title: IL-6–dependent spontaneous proliferation is required for the induction of colitogenic IL-17–producing CD8 + T cells

doi: 10.1084/jem.20071133

Figure Lengend Snippet: Adoptive transfer of naive CD8 + T cells into RAG2 −/− mice causes severe autoimmune colitis. 5 × 10 5 CD44 low CD62L + naive CD8 + T cells from C57BL/6 mice were intravenously injected into RAG2 −/− mice. (A) Body weights of untreated ( n = 5), CD8 + T cell–transferred ( n = 5), or CD4 + T cell–transferred ( n = 5) mice were monitored for 6 wk. Percentages of the resultant body weights against preinjection body weights were calculated every week. The means and SDs are indicated. (B) Colon tissues were obtained from control or CD8 + T cell–transferred mice after 9 wk. Morphology of the representative colon tissues is shown. Bar, 1 cm. (C) HE staining was performed on sections of the colon tissues. Histological pictures of the representative tissues are shown at two different magnifications. Means and SDs of the colitis score are indicated in the bar graph. Bars: (left) 1 mm; (right) 200 μm. (D) Serum KC and SAA levels of untreated or CD8 + T cell–transferred mice were measured by ELISA. The means and SDs are indicated. (E) Cytokine (IFN-γ and IL-17) production by CD8 + T cells from pLNs and mLNs of the adoptively transferred mice 1, 4, and 6 wk after cell transfer. IFN-γ–producing cells in mLNs or pLNs, and IL-17–producing cells in mLNs or pLNs are indicated. Means and SDs of the percentages of cytokine producing cells are shown. (F) Colon sections from untreated or CD8 + T cell–transferred mice were stained with DAPI, anti-CD8 mAb, and anti-CD11b mAb. Bars, 200 μm.

Article Snippet: The cells were passed through a nylon wool column for enrichment of T cells and were stained with FITC-CD44 mAb (IM7; eBioscience), PE-CD62L mAb (MEL-14; eBioscience), and PE-Cy7–CD8a mAb (53-6.7; BD Biosciences).

Techniques: Adoptive Transfer Assay, Injection, Control, Staining, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: IL-6–dependent spontaneous proliferation is required for the induction of colitogenic IL-17–producing CD8 + T cells

doi: 10.1084/jem.20071133

Figure Lengend Snippet: Kinetics of SP essential for inducing pathogenic effector memory T cells in mLNs. 5 × 10 5 cells naive CD8 + T cells from C57BL/6 mice were labeled with CFSE and intravenously injected into RAG2 −/− mice. (A) Proliferation of CD8 + T cells was monitored by flow cytometry 3, 5, and 7 d after the injection. The representative FACS profiles are indicated in the figure. (B) CD44 expression and cytokine production levels of CD8 + T cells from pLNs and mLNs in the adoptively transferred mice were examined by staining with mAbs against CD44, IFN-γ, or IL-17 at day 5. The representative FACS profiles are shown. Percentages are indicated. (C) CFSE-labeled CD8 + T cells from OT-1–TCR transgenic mice were intravenously injected into RAG2 −/− mice. Proliferation of the CD8 + T cells in mLNs was analyzed at day 7. The representative FACS profiles are indicated. (D) CFSE-labeled CD8 + T cells from C57BL/6 mice were intravenously injected into untreated and antibiotic-treated RAG2 −/− mice. Proliferation of the CD8 + T cells in the mLNs of the untreated and antibiotic-treated mice was analyzed by FACS. (E) CFSE-labeled CD8 + T cells from C57BL/6-background Ly5.1 mice were intravenously injected into RAG2 −/− or OT-1/RAG2 −/− mice. Proliferation of the CD8 + T cells in the pLNs or mLNs of OT-1/RAG2 −/− mice was analyzed by FACS.

Article Snippet: The cells were passed through a nylon wool column for enrichment of T cells and were stained with FITC-CD44 mAb (IM7; eBioscience), PE-CD62L mAb (MEL-14; eBioscience), and PE-Cy7–CD8a mAb (53-6.7; BD Biosciences).

Techniques: Labeling, Injection, Flow Cytometry, Expressing, Staining, Transgenic Assay

Journal: Scientific Reports

Article Title: A non-toxic, reversibly released imaging probe for oral cancer that is derived from natural compounds

doi: 10.1038/s41598-021-93408-0

Figure Lengend Snippet: Analysis of the cellular uptake mechanism of compound 1 by SCC-9 cells. ( a ) Inhibition of dynamin related endocytosis and its effect on 1 uptake. Cells were treated with 100 μM Dynasore or 0.1% DMSO as vehicle for 30 min and then co-treated with 5 μM 1 for 1 h at 37 °C and 4 °C. Fluorescence values were corrected for the absorbance at 280 nm. Means ± SD, n = 3. Two-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant. ( b ) Expression of CD44 receptor by SCC-9 cells as analysed by FlowJo Software . A representative histogram, showing the level of expression of CD44 in SCC-9 cells (blue) compared to isotype control (red). ( c ) Titration of the CD44 antibody for its effect on 1 uptake. Cells were treated with different concentrations of CD44 blocking antibody for 30 min and then co-treated with 1 (5 μM) for 1 h. Fluorescence of the cell lysates was corrected against the absorbance at 280 nm. Means ± SD, n = 3. ( d ) Inhibition of CD44 receptor mediated endocytosis of 1 by SCC-9 cells. Cells were treated with 2.5 μg/mL antiCD44 antibody for 30 min and then co-treated with 1 (5 μM) for 1 h at 37 °C and 4 °C. Fluorescence of the cell lysate was corrected against the absorbance at 280 nm. Means ± SD, n = 3. Two-way ANOVA, **** p < 0.0001, n.s. not significant. Graphs were plotted using GraphPad version 6.0c for Mac, www.graphpad.com . ( e ) Inhibition of CD44 receptor mediated endocytosis and colocalization of 1 with the endocytic vesicles. Cells were treated with 2.5 μg/mL CD44 blocking antibody or isotype control antibody for 30 min, then cotreated with 20 μM 1 for 2 h, SYTO Deep Red Nucleic Acid Stain for 1 h, and 30 μg/mL pHrodo Red for 30 min in serum free medium. Cells were then washed twice with PBS and fresh serum free medium added immediately prior to imaging. Bar is 20 μm. Images were analysed using Leica Application Suite X version 3.5.5.19976, www.leica-microsystems.com .

Article Snippet: FITC conjugated IM7 rat monoclonal anti-CD44 antibody and a FITC conjugated rat IgG2b kappa monoclonal isotype control antibody (Invitrogen) were used to quantify CD44 receptor expression in SCC-9, DOK, and MCF-7 cells.

Techniques: Inhibition, Fluorescence, Expressing, Software, Titration, Blocking Assay, Staining, Imaging

Journal: Scientific Reports

Article Title: A non-toxic, reversibly released imaging probe for oral cancer that is derived from natural compounds

doi: 10.1038/s41598-021-93408-0

Figure Lengend Snippet: Uptake of 1 in dysplastic and poorly tumorigenic cells. ( a ) A representative histogram, showing the level of expression of CD44 in DOK (dysplastic, non-tumorigenic) and MCF-7 (poorly tumorigenic) cells analysed by FlowJo Software . 1.5 × 10 5 cells grown in normal culture medium were harvested, washed in PBS, and stained with either anti-CD44 antibody (blue) or isotype control (red) and analysed by flow cytometry. ( b ) A comparative histogram of CD44 expression levels in MCF-7 (orange), DOK (green), and SCC-9 (purple) cells as analysed by FlowJo Software . ( c , d ) Analysis of time-dependent uptake of 1 by SCC-9, DOK, and MCF-7 cells. Cells were seeded on a 6-well plate at 3 × 10 5 cells/well and serum starved for 16 h, and then treated with 5 μM 1 for 24 h, 6 h, 2 h, 1 h, and 30 min at 37 °C, or 4 °C. Cells were then washed twice with PBS and lysed. The fluorescence of the cell lysates was analysed at an excitation of 325 nm and emission of 445 nm. Fluorescence values were corrected for the absorbance at 280 nm. n = 3. Graphs were plotted using GraphPad version 6.0c for Mac, www.graphpad.com .

Article Snippet: FITC conjugated IM7 rat monoclonal anti-CD44 antibody and a FITC conjugated rat IgG2b kappa monoclonal isotype control antibody (Invitrogen) were used to quantify CD44 receptor expression in SCC-9, DOK, and MCF-7 cells.

Techniques: Expressing, Software, Staining, Flow Cytometry, Fluorescence

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) Atg7 gene expression in Atg7 +/+ and Atg7 −/− T cells. T cells were isolated from the spleens of WT and T- Atg7 −/− mice by flow cytometry. mRNA was extracted, and Atg7 gene expression was measured by q-PCR. Bar graph shows relative Atg7 expression in T cells from T- Atg7 −/− mice compared to T cells from WT mice. ****p < 0.0001 by Student's t test. ( B ) Protein was purified from pooled MACS-sorted CD8 + T cells from WT and T- Atg7 −/− mice and western blotted for ATG7. GAPDH was used as a loading control (n = 6 pooled mice). ( C ) LC3 Spot count in WT and Atg7 −/− CD8 + T cells. Splenocytes from WT and T- Atg7 −/− mice were untreated or treated for 2 hr with an autophagy flux inhibitor before staining for relevant cell surface markers and LC3-II. LC3 spot count was assessed using ImageStream software. Quantification is gated on CD8 + T cells. Right panel depicts example LC3 spot count images (×60 magnification). *p = 0.0132, ***p = 0.0002 as determined by Student t test (n = 4) ( D ) Autophagy flux in WT and Atg7 −/− CD8 + T cells. Purified CD8 + T cells were untreated or treated with bafilomycin for 6 hr before whole protein extraction and western blot for LC3. ( E ) Percentage of CD44 hi CD62L − and CD44 hi CD62L + cells in the splenic CD8 + T cell compartment of T- Atg7 −/− and WT mice. Representative FACS plots of three independent experiments are shown, ***p = 0.0003 as determined by Mann–Whitney U-test (n = 9). DOI: http://dx.doi.org/10.7554/eLife.03706.004

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Gene Expression, Isolation, Flow Cytometry, Expressing, Purification, Western Blot, Control, Staining, Software, Protein Extraction, MANN-WHITNEY

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) Frequency of mature single positive CD4 + and CD8 + T cells in thymi of 6 week old mice (n = 4), representative FACS plot of three independent experiments. ( B ) Flow cytometric analysis of CD4 + and CD8 + T cell frequencies in blood and lymph nodes of T- Atg7 −/− and wild-type mice (dot plots depict staining in blood). Quantitative analyses are representative of six independent experiments, *p < 0.05 (n = 4). ( C ) Absolute counts of CD4 + and CD8 + T cells in blood of T- Atg7 −/− and WT mice over time (n = 4). ( D ) Percentage of CD44 hi cells in the splenic CD8 + T cell compartment of T- Atg7 −/− and WT mice. Bar graphs depict the frequency of gated cells (representative of seven independent experiments), *p < 0.05 (n = 4). ( E ) Percentage of splenic CD8 + T cells positive for CD62L in T- Atg7 −/− and WT mice. Data are representative of three independent experiments, *p < 0.05, (n = 4). ( F ) Percentage of CD44 lo and CD44 hi CD8 + T cells expressing Ki-67 in the spleen of T- Atg7 −/− and WT mice. Bar graphs are representative of three independent experiments, *p < 0.05 (n = 4). ( G ) Frequency of splenic CD8 + T cells expressing CD24 in T- Atg7 −/− and WT mice. Bar graph is representative of two independent experiments, *p < 0.05 (n = 4). ( H ) Percentage of donor-derived CD62L + CD8 + T cells. Lethally irradiated CD45.1 hosts reconstituted with a 1:1 mix of T- Atg7 −/− or WT BM (both CD45.2) with CD45.1 wild-type BM. Controls were WT and T- Atg7 −/− mice; *p < 0.05 (n = 4). ( I ) Frequency of donor-derived CD45.2 + CD8 + T cells in the spleen that is CD44 hi . Controls were normal WT and T- Atg7 −/− mice, *p < 0.05 (n = 4). All values are mean ± s.e.m, and all statistical analyses are Mann–Whitney U-tests. DOI: http://dx.doi.org/10.7554/eLife.03706.003

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Staining, Expressing, Derivative Assay, Irradiation, MANN-WHITNEY

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) Flow cytometric analysis of CD4 + and CD8 + T cell frequencies in the spleen of WT and Vav- Atg5 −/− mice. Quantitative analyses are representative of two independent experiments, **p < 0.01 (n = 5). ( B ) Frequency of CD44 hi cells within the CD8 + T cell subset of WT and Vav- Atg5 −/− mice. Data are representative of two independent experiments, **p < 0.01 (n = 5). ( C ) BM reconstitution in BM chimera mice. 9 weeks after marrow transplantation, the frequency of donor CD45.2 cells was assessed in the spleen by flow cytometry. Quantified is the frequency of splenocytes expressing CD45.2 in BM chimera mice and in normal WT and T- Atg7 −/− mice that acted as controls. ( D ) Frequency of CD8 + T cells that are CD45.2 donor-derived in BM chimera mice. Example dot plots are gated on CD8 + T cells. Quantified is the frequency of CD8 + T cells that express CD45.2 in BM chimera and in normal WT and T- Atg7 −/− mice that acted as controls. *p < 0.05 (n = 4). Data are mean ± s.e.m and all statistical analyses are Mann–Whitney U-tests. DOI: http://dx.doi.org/10.7554/eLife.03706.005

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Transplantation Assay, Flow Cytometry, Expressing, Derivative Assay, MANN-WHITNEY

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) Effector CD8 + T cell response to influenza in WT and T- Atg7 −/− mice. Mice were immunized intra-nasally with 0.00032 HAU PR8 influenza. On day 10, antigen-specific CD8 + T cells to nucleoprotein (NP) was assessed with tetramer in lungs. Dot plots show examples of tetramer staining gated on CD8 + T cells. Bar graph indicates percentage of CD8 + T cells specific for NP (n = 5–6) and is representative of three independent experiments. ( B ) Effector CD8 + T cell's response to MCMV in WT and T- Atg7 −/− mice. CD8 + T cells from blood were stained with m45 tetramer on day 7 post-infection. Dot plots show m45-tetramer + cells gated on CD8 + T cells. Bar graph indicates % CD8 + T cells specific for m45 (n = 4–5) and is representative of three independent experiments. ( C ) CD8 + T mem response to influenza. WT and T- Atg7 −/− mice immunized as in ( A ) and the antigen-specific CD8 + T cell response was assessed in lungs on day 50 by tetramer. *p < 0.05, by Mann–Whitney U-test (n = 4). Dot plots are gated on CD8 + T cells. Bar graph is representative of three independent experiments. ( D ) CD8 + T mem 's response to MCMV. Lung CD8 + T cells on day 65 post-infection were stained with IE3-tetramer. Dot plots are gated on CD8 + T cells. Quantitation depicts frequency of IE3 specific CD8 + T cells. *p < 0.05, by Mann–Whitney U-test (n = 4). Data are representative of two independent experiments. ( E ) CD8 + T cell kinetics to influenza infection. WT and T- Atg7 −/− mice were immunized as in ( A ) and CD8 + T cell response tracked over time in blood by tetramer. Y-axis shows frequency of NP-specific CD8 + T cells. ( F ) CD8 + T cell kinetics to MCMV infection. CD8 + T cell response to epitopes m38 (left panel) and IE3 (right panel) were tracked over time in blood by tetramer in WT and T- Atg7 −/− mice. Y-axis indicates the percentage of CD8 + T cells that are m38-specific. ( G ) Influenza virus titres. WT and T- Atg7 −/− mice were culled at days 3 and 6 post-immunization with PR8, and lungs were collected and snap frozen in liquid nitrogen. Virus titres were determined using MDCK-SIAT1 cells. ***p = 0.0002 as determined by Student t-test (n = 4) ( H ) WT mice were immunized with PR8 influenza as in ( A ). CD8 + T cells from spleen were stained with CytoID at day 9 post-infection and assessed by flow cytometry. Histograms show examples of CD44 lo CD8 + T cells from unimmunized mice (filled grey line) and in NP-specific CD8 + T cells from immunized mice (open black line). Quantification is by mean fluorescence intensity (MFI) of CytoID on gated indicated cell population and representative of two independent experiments. ****p < 0.0001 by Student t test (n = 6). ( I ) Autophagy levels by CytoID staining on CD8 + T cells from the lungs on day 9 post-infection as in ( G ). Histograms provide examples of CytoID staining, gated on CD8 + T cells in lungs of immunized mice (solid grey lines), and gated on NP-tetramer-specific CD8 + T cells from immunized mice (open black lines). Quantification is by mean fluorescence intensity (MFI) of CytoID on gated indicated cell population and representative of two independent experiments. ****p < 0.0001 by Student t test (n = 4–8). All values are mean ± s.e.m. DOI: http://dx.doi.org/10.7554/eLife.03706.006

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Staining, Infection, MANN-WHITNEY, Quantitation Assay, Virus, Flow Cytometry, Fluorescence

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) CD8 + T mem response to influenza in mixed BM chimeras, generated as in . Mice were immunized with PR8 influenza, and the CD8 + T mem response of the CD45.2 donor to NP was assessed in lungs by tetramer on day 40. Quantitation shows frequency of (donor) CD45.2 + CD8 + T cells that are NP-specific (n = 5–6). **p < 0.01, by Mann–Whitney U-test (n = 4–7). ( B ) SLEC and MPEC populations in the Atg7 +/+ and Atg7 −/− antigen-specific CD8 + T cell pool. Mixed BM chimera generated as in , were immunized with MCMV 8 weeks after transplantation. Dot plots show example of KLRG1 and CD127 expression on gated CD45.2 + m45-tetramer + CD8 + T cells on day 10 post-infection. Upper bar graph depicts the % of CD45.2 + m45-tetramer + CD8 + T cells that are CD127 − KLRG1 + (SLECs). Lower bar graph shows the % of CD127 + KLRG1 − (MPECs) in the same population. *p < 0.05, by Mann–Whitney U-test (n = 4–7). ( C ) Markers of exhaustion on Atg7 −/− MCMV-specific CD8 + T cells on MCMV challenged BM chimera. Dot plots depict example of PD-1 and TIM-3 staining on gated CD45.2 + m45-tetramer + CD8 + T cells. Bar graph quantifies the percentage of (donor) CD45.2 + m45-tetramer + CD8 + T cells that are PD-1 + TIM-3 + at day 10 post-infection. *p < 0.05, by Mann–Whitney U-test (n = 4–7). ( D ) CD127 expression on Atg7 −/− MCMV-specific CD8 + T cells in MCMV challenged BM chimeras. Examples of CD127 staining on gated CD45.2 + m45-tetramer + CD8 + T cells from spleen on day 10 post-infection are shown. *p < 0.05, by Mann–Whitney U-test (n = 4–7). ( E ) IL-15Rα expression on splenic Atg7 −/− MCMV-specific CD8 + T cells in MCMV challenged BM chimeras. Histograms depict IL-15Rα expression in CD44 lo CD8 + T cells from unimmunized mice (grey dotted line), Atg7 +/+ CD45.2 + m45-tetramer CD8 + T cells (grey filled line), and Atg7 −/− CD45.2 + m45-tetramer CD8 + T cells (black line). The left histogram shows expression in control normal WT and T- Atg7 −/− mice, the right histogram indicates staining in donor CD45.2 + cells from BM chimera mice. Quantified is IL-15Rα mean fluorescence intensity on gated CD45.2 + m45-tetramer CD8 + T cells (n = 4–7). All values are mean ± s.e.m. DOI: http://dx.doi.org/10.7554/eLife.03706.008

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Generated, Quantitation Assay, MANN-WHITNEY, Transplantation Assay, Expressing, Infection, Staining, Control, Fluorescence

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) IRF4 expression in Atg7 −/− and Atg7 +/+ splenic m45-specific CD8 + T cells on day 9, 15, and 22 post-infection. As a control, IRF4 was also measured in CD44 lo CD8 + T cells from unimmunized mice (naïve). Quantification shows IRF4 mean fluorescence intensity from gated m45-tetramer + CD8 + T cells and CD44 lo CD8 + T cells (naïve). Statistics—Student's t test (n = 4–5). ( B ) EOMES expression in Atg7 −/− and Atg7 +/+ antigen-specific CD8 + T cells. WT and T- Atg7 −/− mice were immunized with MCMV and EOMES expression was measured in m45-specific CD8 + T cells on day 9, 15, and 22. As a control, EOMES was also measured in CD44 lo CD8 + T cells from unimmunized mice (naïve). Quantification shows EOMES mean fluorescence intensity on gated m45-tetramer + CD8 + T cells and CD44 lo CD8 + T cells (naïve) (n = 4–5). DOI: http://dx.doi.org/10.7554/eLife.03706.009

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Expressing, Infection, Control, Fluorescence

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) The spleens of unimmunized and MCMV-infected mice were stained with the apoptotic marker Annexin V and a dead cell dye that stains cells with disrupted membranes on the time points indicated. Apoptotic cells were defined as dead cell dye-negative Annexin V + . Dot plots are gated on either CD44 lo CD8 + T cells (naïve) or m45-tetramer + CD8 + T cells. *p < 0.05, by Mann–Whitney U-test (n = 4–5). ( B ) Mitochondrial volume by MitoTracker Green in Tetramer + CD8 + T cells from WT and T- Atg7 −/− mice. Spleens from MCMV-immunized mice were stained with MitoTracker Green on day 15 post-infection. Quantification depicts mean fluorescence intensity on m45-tetramer + CD8 + T cells and is representative of three independent experiments. **p < 0.01, Student t test (n = 4–5). ( C ) Mitochondrial superoxide production in Tetramer + CD8 + T cells by MitoSox. Spleens from MCMV-immunized WT and T- Atg7 −/− mice were stained with MitoSox Red on day 15 post-infection and analyzed by flow cytometry. Bar graph depicts mean fluorescence intensity on m45-tetramer + CD8 + T cells and is representative of three independent experiments. **p < 0.01, by Student t test (n = 4–5). ( D ) GLUT-1 expression. Tetramer + CD8 + T cells from MCMV-immunized WT and T- Atg7 −/− mice were stained with the GFP-tagged HTLV receptor binding domain (eGFP-H RBD ), that binds GLUT-1, at the time points indicated. As a control, GLUT-1 was also measured on CD44 lo CD8 + T cells from unimmunized mice (naïve). Bar graph shows the percentage of cells expressing GLUT-1. *p < 0.05, as determined by Mann–Whitney U-test (n = 4–5). All values are mean ± s.e.m. DOI: http://dx.doi.org/10.7554/eLife.03706.010

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Infection, Staining, Marker, MANN-WHITNEY, Fluorescence, Flow Cytometry, Expressing, Binding Assay, Control

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) MCMV-immunized WT and T- Atg7 −/− mice were assessed for Bcl-2 expression in splenic m45-specific CD8 + T cells on day 9 and 22 post-infection. As a control, Bcl-2 was measured in CD44 lo CD8 + T cells from unimmunized mice (naïve). Quantification shows Bcl-2 mean fluorescence intensity. Statistics—Student's t test (n = 4–5). ( B ) GLUT-1 antibody staining on day 9 and day 22 post-infection in MCMV-immunized WT and T- Atg7 −/− mice. As a control, GLUT-1 was assessed in CD44 lo CD8 + T cells from unimmunized mice (naïve). Bar graphs indicate the frequency of m45-tetramer + CD8 + T cells and CD44 lo CD8 + T cells (naïve) that express GLUT-1. Statistics—Mann Whitney U-test (n = 4–5). DOI: http://dx.doi.org/10.7554/eLife.03706.011

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Expressing, Infection, Control, Fluorescence, Staining, MANN-WHITNEY

Journal: eLife

Article Title: Autophagy is a critical regulator of memory CD8 + T cell formation

doi: 10.7554/eLife.03706

Figure Lengend Snippet: ( A ) Autophagy gene expression in CD8 + T cells from young and elderly mice. CD44 lo and CD44 hi CD8 + T cells were purified from 6 week old and 2 year old mice using fluorescent activated cell sorting. mRNA was extracted and the expression of essential autophagy genes was measured by q-PCR. Shown is the fold change in expression in CD8 + T cells from old mice relative to expression in young mice (normalized to gapdh and hprt ). ( B ) LC3 Spot count in CD8 + T cells from young and old mice. Splenic CD8 + T cells from 8 week old and 2 year old mice were treated with an autophagy flux inhibitor for 2 hr, as a control cells were left untreated. LC3 spot count was determined on CD8 + CD44 hi T cells using ImageStream. Representative images are shown (×60 magnification). **p = 0.0082, ***p = 0.0004 as determined by Student t-test (n = 4–5). ( C ) Quantification for images shown in ( B ). ( D ) Human T cell line Jurkat was incubated with 100 µM spermidine for 2, 4, or 6 hr or left untreated followed by whole protein extraction for LC3 Western Blot (upper panel). In the lower panel, Jurkat cells were incubated either with no spermidine (control), 100 µM, or 500 µM spermidine for 6 hr and then Western blotted for LC3. GAPDH was used as loading control for all Western blots. ( E ) Jurkat cells were treated with 1 mM DFMO or 1 mM DFMO with 1 µM spermidine for 48 hr or left untreated (control). In the final 6 hr of incubation, all cells were treated with 10 nM bafilomycin A1 and LC3-I to LC3-II conversion was assessed by Western Blot. ( F ) Jurkat cells were treated with 50 nM rapamycin, 100 µM or 500 µM spermidine for 6 hr followed by detection of phosphorylated S6 (Ser235/236) by Western Blot. As a control, cells were left untreated. ( G ) CD8 + T cell kinetics to influenza vaccination in aged mice in the presence of spermidine. 8-week-old young WT and 23 month old WT and T- Atg7 −/− mice were vaccinated 22 days apart with S-Flu. 21 days prior to the first vaccination, aged WT and T- Atg7 −/− mice were administered spermidine in the drinking water at a concentration of 5 mM through to the experimental endpoint. As a control, 23 month old WT mice were administered water alone. The CD8 + T cell response to NP was tracked over time in the blood by tetramer (n = 4–5). Y-axis depicts the frequency of CD8 + T cells that are specific for NP. **p < 0.01 by Mann–Whitney U-test. ( H ) CD8 + T cell kinetics to influenza vaccination and challenge in aged mice in the presence of spermidine. 8-week-old young WT and T- Atg7 −/− and 23 month old WT and T- Atg7 −/− mice were vaccinated as described in ( G ). 30 days after the last vaccination, mice were challenged with 32 HAU X31 and the CD8 + T cell response to challenge was measured 9 days later in the lungs by tetramer. From 21 days prior to the first vaccination, through to the experimental end point, aged WT and T- Atg7 −/− mice were administered spermidine as before. Y-axis indicated the percentage of CD8 + T cells that are specific for NP (n = 4–5). ( I ) CD8 + T cell response to influenza challenge in vaccinated aged mice in the presence of spermidine. 9 days post–challenge, lungs were harvested and the NP-specific CD8 + T cell response to influenza challenge was measured by tetramer. Example dot plots are gated on CD8 + T cells. Bar chart shows absolute counts of NP-specific CD8 + T cells in the lung per lobe. **p < 0.01, ****p < 0.0001 by Student t test (n = 4–5). All values are mean ± s.e.m. DOI: http://dx.doi.org/10.7554/eLife.03706.014

Article Snippet: The following antibodies were used for flow cytometry (antibody clone in brackets): CD8 (Ly2) PE/PE-CY7; CD8 (53-6.7) FITC/eF450/PE-Cy7; CD4 (GK1.5) PE/FITC/APC; TCRβ (H57-597) FITC/PE/PE-Cy7/APC; CD3 (145-2C11) eF450/APC; CD62L (MEL-14) FITC/PE-Cy7; CD44 (IM7) FITC/PE-Cy7; KLRG1 (2F1) FITC/PE-Cy7; IRF4 (3E4) FITC; EOMES (Dan11mag) PE-Cy7; CD127 (A7R34) FITC/PE/eF450; Ki-67 (SolA15) eF450; CD24 (M1/69) APC; CD16/32 (93) purified Fc block; CD45.2 (104) eF450; all from eBioscience (San Diego, CA, USA).

Techniques: Gene Expression, Purification, FACS, Expressing, Control, Incubation, Protein Extraction, Western Blot, Concentration Assay, MANN-WHITNEY