CD4 and CD8 T cells express similar levels of CD3 and IL-2 receptor.

<p>Isolated CD4 and CD8 T cells were stimulated on anti-CD3 anti-CD28 coated plates in the presence of IL-2 for the times shown. Surface expression of CD3, CD28, CD25, CD132 and CD122 were measured by flow cytometry. Data are representative from two independent experiments.</p

CD4 T cells are more oxidative than CD8 T cells.

<p>CD4 and CD8 T cells were activated for 48 or 72-CD3 anti-CD28 and then (<b>A</b>) oxygen consumption rate (OCR) and (<b>B</b>) extracellular acidification rate (ECAR) were measured in comparison to CD4 and CD8 T cells isolated directly <i>ex vivo.</i> Cells were activated in regular culture media, and flux analysis was performed in media containing 10 mM D-glucose, 10 mM L-glutamine, and 10 mM sodium pyruvate. (<b>C</b>) OCR/ECAR ratio was calculated for 48 and 72 h stimulated cells. Data show mean ± standard deviation from representative experiment. Data are representative from a minimum of two independent experiments.</p

Activation context fine-tunes metabolic reprogramming.

<p>(A, B) Isolated CD4 and CD8 T cells were stimulated for 72 h on plates coated with high (10 µg/ml), medium (2.5 µg/ml) or low (0.5 µg/ml) doses of anti-CD3 and anti-CD28, plus 20 ng/ml IL-2, 10 ng/ml IL-15, or 10 ng/ml IL-7 as indicated. Shown are (<b>A</b>) mitotracker staining of mitochondrial mass, and (<b>B</b>) DCFDA measurement of reactive oxygen species. (<b>C</b>) Isolated CD4 and CD8 T cells were stimulated for 72 h on plates coated with 10 µg/ml anti-CD3, anti-CD28, anti-41BB, or anti OX40, as indicated. Mitotracker staining was quantified by flow cytometry. (<b>A–C</b>) All data are representative of a minimum of two experiments.</p

CD8 T cell growth is more resistant than CD4 T cell growth to glycolytic inhibition.

<p>CD4 and CD8 T cells were labeled with CellTrace Violet and activated for 48-CD3 anti-CD28 in the presence of indicated doses of rotenone or 2-deoxyglucose (2-DG). (<b>A, B</b>) Mean forward light scatter (cell size) of activated cells and (<b>C, D</b>) expansion index, (the fold expansion of the original culture) as determined by CellTrace violet dilution are shown. Data shown are representative of two experiments; data show mean ± standard deviation.</p

CD4 T cells have greater mitochondrial content and reactive oxygen species than CD8 T cells.

<p>CD4 and CD8 T cells were activated for 72-CD3 anti-CD28. CD4 and CD8 cells were isolated directly <i>ex vivo</i> for comparison and then cells were (<b>A</b>) lysed and analyzed by immunoblot mitochondrial OXPHOS complex subunits; or (<b>B</b>) stained with mitotracker to measure mitochondrial content, TMRE, to measure mitochondrial membrane potential, or DCF, to measure reactive oxygen species. Mitotracker and DCF staining were quantified by flow cytometry and histograms are overlaid as indicated. (<b>A</b>) CD4+CD25+ depletion was not performed prior to stimulation. (<b>A–B</b>) All data are representative of a minimum of three experiments.</p

Following activation CD4 and CD8 T cells undergo similar glycolytic reprogramming events.

<p>CD4 and CD8 T cells were activated for 72-CD3 anti-CD28 in the presence of IL-2. CD4 and CD8 cells were isolated directly <i>ex vivo</i> for comparison and then all cells were lysed and analyzed by immunoblot for expression of (<b>A</b>) Glut1, or (<b>C</b>) hexokinase, PKM2, cytochrome C, Ser235/236 phosphorylated small ribosomal subunit S6, and total S6. (<b>B</b>) CD4 and CD8 Glut1^myc/myc T cells expressing myc tagged Glut1 at endogenous levels were isolated and activated for 48 h with anti-CD3 anti-CD28 in the absence of IL-2. Surface expression of Glut1^myc was then measured by flow cytometry. Glut1^myc/myc T cells analyzed directly <i>ex vivo</i> were used as a control. (<b>A–C</b>) CD4+CD25+ depletion was not performed prior to stimulation. All data are representative of a minimum of three independent experiments.</p

CD8 T cells proliferate faster than CD4 T cells.

<p><b>(A)</b> CD4 and CD8 T cells were stimulated with anti-CD3 anti-CD28 and the number of viable cells was counted as shown. (<b>B</b>) CD4 and CD8 T cells were labeled with CellTrace Violet and then activated with anti-CD3 anti-CD28 for 3 days. CellTrace Violet dilution was measured by flow cytometry. Gates show generation numbers. (<b>C</b>) CD4 and CD8 T cells were activated for 3 days with anti-CD3 anti-CD28 and propidium iodide staining was then used to detect cellular DNA content by flow cytometry. Small gates show percentage of non-doublet cells with sub-diploid DNA content, large gates show percentage of non-doublet cells in G₂ and S cycle phase. (<b>A–C</b>) All data are representative from a minimum of (<b>a</b>) two or (<b>b</b>, <b>c</b>) three independent experiments.</p

CD4 T cells have a higher respiratory capacity and are more oxidative than CD8 T cells.

<p><b>(A, B)</b> CD4 and CD8 T cells were activated for 72-CD3 anti-CD28 and then compared to CD4 and CD8 T cells isolated directly <i>ex vivo</i>. (<b>A</b>) Real time changes in oxygen consumption rate (OCR) were measured in response to addition of the indicated compounds. OCR analysis was performed in media containing 10 mM D-glucose, 10 mM L-glutamine, and 10 mM sodium pyruvate. (<b>B</b>) CD4 and CD8 T cells were activated for 72 h with anti-CD3 anti-CD28 and then OCR was determined by extracellular flux analysis. Cells were activated in regular culture media, and flux analysis was performed in media supplemented with 10 mM D-glucose, 10 mM L-glutamine, or 10 mM sodium pyruvate as indicated. (<b>A–C</b>) Data shown are representative mean ± standard deviation from two independent experiments.</p

CD8 T cells grow more rapidly than CD4 T cells.

<p>Isolated CD4 and CD8 T cells were activated with anti-CD3 anti-CD28 and median cell volume in fl was measured by Coulter counter at the timepoints indicated. Data show median ± standard deviation of technical triplicates. Data shown are representative from three independent experiments.</p

OCT4 and Sox2 gene expression in ovarian cancer cell lines.

<p>OCT4 and Sox2 gene expression in ovarian cancer cell lines.</p