Stalled ribosomes in resting ex vivo human lymphocytes

(A) Primary human lymphocytes from three independent donors were cultured in PMA/ionomycin and IL-2 (+PMA/iono) or IL-2 only (-PMA/iono) for up to 5 days. CD45+ cells were processed for flow RPM. (B) Primary human lymphocytes were cultured ex vivo as indicated, followed by a 15 minute treatment with vehicle, harringtonine (HAR, 5μg/mL), pactamycin (PA, 10μM), emetine (EME, 25μg/mL), or cycloheximide (CHX, 200μg/ml), and all cultures were then treated with puromycin (PMY, 50µg/mL) for 5 minutes. Cells were harvested, and RPM staining was performed. Gated on CD45+ cells. Error bars represent standard deviation of two independent experiments. (C) Radioactive amino acid incorporation (0.2 mCi/mL [3H]-Leu for 5 min) or RPM (as in B) in day 1 non-activated human lymphocytes. Error bars represent standard deviation of two independent experiments. (D) Radioactive amino acid incorporation and RPM in rested and activated human lymphocytes. RPM MFI values (gated on CD45+ cells) on the left, [3H]-Leu incorporation (cpm) in the middle, and ratios of the activated to the resting cells on the right. Each point represents a single donor; bars indicate the mean from 3-5 independent donors. Left and middle panels: one-way ANOVA pairwise p-values; right panel: unpaired t-test p-values with Welch’s correction.

RPM measures ribosome transit times in HeLa and human lymphocytes

(A) Schematic representation of the ribopuromycylation (RPM) Ribosome Transit Analysis (RTA) method. Translation initiation is blocked and the decrease in RPM is monitored as the elongating ribosomes run off mRNA. (B) RPM-RTA in HeLa cells. Harringtonine (HAR, 5μg/mL) is used to inhibit new ribosome initiation; emetine (EME, 25μg/mL) is used to freeze ribosomes on mRNA; puromycin (PMY, 50μg/mL) generates RPM signal. Curve is fitted using one phase exponential decay, and ribosome transit times are expressed as RPM half-time to decay. (C) Same as B, but cells are instead lysed in the presence of MG-132 and subjected to anti-puromycin western blot analysis. (D) Representative plots of the RPM-RTA signal in resting and activated human lymphocytes (left three panels). Gated on CD45+ cells. Far right, ribosome transit times determined from 3 independent donors. Each dot represents data from one individual donor; the horizontal bars indicate the mean. P-values indicate one-way ANOVA pairwise comparisons. (E) Ribosome transit times as in A but determined by [3H]-Leu incorporation instead of RPM. After treatment with HAR or HAR plus EME, cells were labeled for 5 minutes in 0.25mCi/mL [3H]-Leu. Right panel, ribosome transit times determined by [3H]-Leu incorporation from three independent donors. Each dot represents data from one individual donor; the horizontal bars indicate the mean. Unpaired t-test.

RPM ribosome transit analysis of OT-I T cells in vitro

(A) Lymphocytes from spleens and lymph nodes from transgenic OT-I mice were isolated, and either used immediately, cultured for one day in the absence of PMA/ionomycin, or cultured for 2 days in the presence of PMA/ionomycin and IL-2. RPM-RTA analysis was conducted to determine ribosome transit half-lives, both with and without EME. (B) Lymphocytes from spleens and lymph nodes from transgenic OT-I mice were isolated, labeled with CFSE, and cultured under activating conditions for either 24 or 48 hours. Cells were harvested, and RPM-RTA was performed at both 37°C and 39.5 °C. Half-life of RPM signal by RTA is plotted; p-values determined by paired t-test analysis.

Translation rates of resting and activated T cells in vivo

(A) Depiction of the in vivo RPM-RTA method. Labeled OT-I T cells are first adoptively transferred, followed by VACV-SIINFEKL infection of mice. RTA analysis is performed by intravenous injection of HAR followed by PMY (+/- CHX to prevent leakiness from HAR inhibition alone). Spleens are harvested for RPM analysis on both endogenous and transferred T cells. Schematic designed with Biorender. (B) CFSE-labeled Ly5.2+ (CD45.2+CD45.1-) OT-I T cells were adoptively transferred into Ly5.1 (CD45.1+CD45.2-) mice, which were then infected with VACV-SIINFEKL to activate the OT-I cells. Three days after infection, mice were intravenously injected with HAR simultaneously with PMY for 5 minutes (maximum signal), or first injected with HAR for ∼110, ∼275, or ∼575 seconds before being injected with PMY for 5 minutes. Splenocytes from mice were harvested, surface stained for gating and activation markers as indicated, fixed and permeabilized, and stained for RPM. Gates were CFSElow OT-I CD8+ T cells to measure decay in activated cells, and CD44-CD8+ or CD44-CD4+ T cells to measure decay in resting T cells. The curve was generated by fitting to a one phase exponential decay. Representative of two independent experiments, 2-4 mice per group, with the mean and standard deviation of the calculated half-life decays as indicated. (C) RTA, with the CHX modification, of adoptively transferred OT-I T cells or un-activated host CD8+ T cells in mice infected for 2 or 3 days with VACV-SIINFEKL. 3-4 independent experiments combined, normalized by setting maximum background-subtracted signal to 100.

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Puromycylation reveals percentage of actively translating monosomes in resting and activated T cells

(A) OT-I mice were treated intravenously with CHX and PMY, and lymphocytes from the spleens and lymph nodes were isolated and subjected to polysome profiling by ultracentrifugation through 15-45% sucrose gradients (resting OT-I T cells). OT-I T cells activated in vitro for 2 days with PMA/ionomycin and IL-2 (without cognate SIINFEKL peptide) were treated either with CHX alone (no PMY control) or CHX with PMY and subjected to polysome profiling. The indicated fractions were collected, pooled, and their ribosomes were re-isolated and dotted onto a nitrocellulose membrane for blotting with antibodies against PMY and RPL7. After subtraction of background signal from the anti-puro antibody (middle panel), the PMY/RPL7 ratio of monosomes was expressed relative to that of polysomes, which was defined as 100% translating. Representative of two independent experiments. (B) For resting T cells, OT-I mice were treated intravenously with CHX, and lymphocytes from the spleens or lymph nodes were isolated and lysed. For activated T cells, lymph node or splenic OT-I T cells were stimulated in vitro for 2 days with PMA/ionomycin, IL-2, and exogenous SIINFEKL, followed by treatment with CHX for 5 minutes. For both resting and activated cells, ribosome-containing lysates were fractionated via ultracentrifugation on 15-45% sucrose gradients.

T cell accounting reveals discrepancy in proteome duplication rate for activated T cells in vivo

(A) Measurements made to calculate in vitro and in vivo rates of T cell division. (B) Volume calculations based on diameter measurements made by automated cell counter for the indicated cell types. Day 1 and day 2 represent in vitro activated OT-I T cells. Ex vivo day 2 represent cells activated in vivo for 2 days, followed by isolation and processing. (C) Protein content per cell as measured by tryptophan fluorescence of denatured lysates. (D) Protein molecules per fL, assuming an average protein length of 472 aa and average amino acid mass of 110 Da. (E) Example output from custom bioanalyzer method to determine number or ribosomes per cell. Total RNA is quantified and the bioanalyzer is used to determine area under the curve for 18S and 28S percentage of total RNA. Additionally, an exogenous mRNA standard is spiked into the sample prior to RNA isolation to determine the percent loss in yield during the purification procedure. Combined, this method allows for the accurate determination of total number of 18S and 28S molecules per cell. (F) Number of ribosomes per cell for the indicated cells. (G) Ribosome per fL for the indicated cells. (H) The protein/ribosome ratio, a representation of how many proteins a single ribosome would need to create to duplicate the proteome. (I) Discrepancy between measured and calculated rates of division for OT-I T cells activated and dividing in vivo.