Stalled ribosomes in resting ex vivo human lymphocytes

(A) Primary human lymphocytes from three independent donors were cultured in PMA/ionomycin and IL2 (+PMA/I) or IL-2 only (-PMA/I) 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, harringtonin (HAR, 5μg/mL), pactamycin (PA, 10μM), emetine (EME, 25μg/mL), or cycloheximide (CHX, 200μg/ml). Cells were harvested, and surface 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 resting human lymphocytes. Error bars represent standard deviation of two independent experiments. (D) Radioactive amino acid incorporation and RPM in resting and activated human lymphocytes. RPM MFI values (gated on CD45+ cells) on the left, [3H]-Leu incorporation values (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.

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 by flow cytometry as the elongating ribosomes run off mRNA. (B) RPM-RTA in HeLa cells. Harringtonin (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 CD45+ lymphocytes (left three panels). Far right, ribosome transit times determined from 3 independent donors. Each dot represents data from one individual donor; the horizontal bars indicate the mean. (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.

RPM ribosome transit analysis in 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 IL2. 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 in the presence of IL-2 and PMA/ionomycin (+PI) or SIINFEKL, PMA/ionomycin, and IL2 (SIIN+PI) for either 24 or 48 hours. Cells were harvested, and RPM-RTA was performed at both 37ºC and 39.5 ºC. 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 RPM-RTA in vivo method. Labeled OT-I T cells are first adoptively transferred, followed by VACV-SIINFEKL infection. RTA analysis is performed by intravenous injection of HAR and PMY (+/-CHX to prevent leakiness from HAR inhibition alone). Spleens are harvested for RPM analysis on both endogenous and transferred T cells. (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 CFSElo 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 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.

Puromycylation reveals 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 T cells). OT-I T cells activated in vitro for 2 days with PMA/ionomycin and IL2 (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-PMY antibody (middle panel), the PMY/RPL7 ratio of monosomes was expressed relative to that of polysomes, which we define as 100% translating. Representative of two independent experiments. (B) For resting T cells, OT-I mice were treated IV 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, IL2, 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) Diameter measurements made by automated cell counter for the indicated cells. Day 1 and Day 2 represent in vitro activated OT-I T cells. (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 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 purification to determine the percent loss in yield during the purification procedure. Combined, these methods allow for the accurate determination of total number of 18S and 28S molecules per cell. (F) 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.

RPM tracks translation in distinct cell populations over time

(A) Population frequency and RPM of resting D2 or D5 human lymphocytes or PMA/ionomcyin and IL2 activated D2 or D5 human lymphocytes. Left panel is the percent of CD45+ cells in the indicated population, the right panel is the amount of RPM signal in each population. (B) Representative RPM flow cytometry plot gated on CD8+ T cellsSimilar data was obtained from all donors.

Dominant populations of monosomes in resting human and mouse lymphocytes

(A) Primary human lymphocytes were cultured for one day in the absence of PMA/ionomycin or for up to 2 days in the presence of PMA/ionomycin and IL2, and the cells were subjected to polysome profiling. Representative of two independent experiments. (B) Freshly isolated human lymphocytes were treated with 0.1μg/mL CHX for 30 minutes prior to cell lysis and sucrose gradient centrifugation. Representative of two independent experiments. (C) C57BL/6 mice were treated IV with vehicle or CHX. After 10 min, spleens and lymph nodes were harvested, and the resulting cells subjected to polysome profiling via ultracentrifugation through 15-45% sucrose gradients. (D) Lymphocytes or hepatocytes were harvested from OT-I mice treated IV with CHX, lysed, and processed for polysome profiling. For activated cells, OT-I T cells were treated with PMA/ionomycin and IL2 for 2 days prior to CHX treatment and polysome profiling. Bottom right; quantification of the areas under the curve of free subunits, monosomes, and polysomes. Representative of two independent experiments.

RPM cell phenotyping

(A) C57BL/6 mice were treated intravenously with CHX and PMY or only PMY. After the indicated times, splenocytes were harvested, surface stained, fixed/permeabilized, and RPM staining was performed. Representative of three independent experiments, 2-3 mice per group. (B) In one set of C57BL/6 mice, HAR was intravenously injected for 15 minutes before intravenously injecting mice with PMY for 5 min. In a second set of mice, CHX and PMY were IV injected for 5 min. Splenocytes from each set of mice were harvested, surface stained, fixed, and permeabilized, and RPM staining was performed for various immune cell subsets. To determine relative amounts of ribosomes, the ‘HAR then PMY’ RPM signal was subtracted from the CHX+PMY RPM signal for each cell subset after flow cytometry. Representative of two independent experiments, 2-4 mice per group. (C) CFSE-labeled Ly5.2+ (CD45.2+CD45.1-) OT-I cells were adoptively transferred into Ly5.1+ (CD45.1+CD45.2-) mice, which were then infected with VAC-SIINFEKL. 1-3 days after infection, mice were intravenously injected with CHX simultaneously with PMY for 5 min. Splenocytes from the mice were harvested, surface stained, fixed and permeabilized, and RPM staining was performed. Representative flow cytometry plots gated on OT-I T cells. (D) Number of divisions (by CFSE dilution) of OT-I T cells 1-3 days after infection of mice with VACV-SIINFEKL. (E) Amount of translation as measured by RPM signal (with no,PMY signal subtracted) in uninfected, or one-, two-, or three-day VACV-SIINFEKL-infected mice. Representative of four independent experiments, 2-3 mice per time point.

Exogenous SIINFEKL significantly enhances OT-I T cell activation in vitro

(A) Maximum-normalized MFI of CD69, CD25, CD44, and side scatter (SSCa), as well as CFSE expansion index, of OT-I T cells after 1 or 2 days with PMA/ionomycin or PMA/ionomycin with exogenous SIINFEKL. IL-2 was included in all conditions. (B) Side scatter is a good proxy for cell size. SSCa MFI plotted vs cell diameter as determined by automated cell counter measurements.

Salt stability of T cell ribosomes and monosome quantification

(A) LN and splenic OT-I T cells were mixed and stimulated in vitro for 2 days with SIINFEKL, PMA/ionomycin, and IL2. Cells were treated with CHX for 5 minutes, lysed, and brought to either 300 or 500mM NaCl final concentration. Ribosome-containing lysates were subjected to polysome profiling via ultracentrifugation through 15-45% sucrose gradients containing either 300 or 500mM NaCl. (B) Quantification of the areas under the curve of free subunits, monosomes, and polysomes for each sample. (C) For resting T cells, OT-I mice were treated IV with CHX and lymphocytes from the spleens or LN were isolated and lysed. For activated T cells, LN and splenic OT-I T cells were mixed and stimulated in vitro for 2 days with SIINFEKL, PMA/ionomycin, and IL2, treated with CHX for 5 minutes, and lysed. Lysates were subjected to polysome profiling via ultracentrifugation on 15-45% sucrose gradients after bringing both lysate and sucrose gradients to a final concentration of 500mM NaCl to dissociate non-translating ribosomes. (D) Quantification of the areas under the curve of free subunits, monosomes, and polysomes in each sample. Representative of two independent experiments.

Fractionation of HeLa or T cells reveals few ribosomal components in nuclear lysates

HeLa cells, freshly isolated resting OT-I T cells, or OT-I T cells stimulated with PMA/ionomycin and IL2 in vitro for 2 days were either lysed directly in SDS extraction buffer (“all”) or subjected to a hypotonic lysis procedure to isolate non-nuclear lysates and nuclear lysates. Equal amounts of each fraction were subjected to immunoblotting for markers typical of the cytosol, ER, and nucleus. Antibodies against ribosomal proteins were used to determine where the majority of ribosomal proteins (and therefore ribosomes) fractionated. Controls with Abs specific for nucleolar located fibrillarin, histone H3, lamin A/C establish lack of nuclear contamination of non-nuclear fraction and with cytoplasmic located HSP90, GRP94, PDI, actin lack of cyotoplasmic contamination of nuclear fraction.