PMC

Levels of nonautonomous miRNAs from deep sequencing under different normalization conditions. (A) Expression levels of EBV-BHRF-1/2 from CD3⁺ cells after a 3-h incubation with Raji cells. The red line denotes the normalized expression level of EBV-BHRF-1/2 found in the incubated CD3⁺ cells. The histogram shows the expected distribution of EBV-BHRF-1/2 that would be induced by 0.5% Raji contamination as calculated using bootstrapping. The blue line indicates the mean value of this distribution. The difference in standard deviations bounds the type I error. We analyzed this effect with five different normalization procedures, and reported the most conservative results: P < 0.015 (normalizations to tRNA levels). (B) Endogenous miR transfer. Expression levels of the miR-18a from deep sequencing under normalization conditions as in A. The most conservative results are normalization to miR-31 that corresponds to P < 0.0004.

Nonautonomously expressed shRNAs function in recipient T cells. (A) A schematic presentation of the experiment depicts donor Raji cells stably expressing an shRNA against luciferase, a CD4 shRNA, or a control mouse p53 shRNA. These cells were cocultured either with Jurkat-3′-luciferase-Venus cells or with normal human CD4⁺ T cells. Decreases in the relevant target sensor (luciferase, detected by the venus fluorophore or CD4, detected by red fluorescent mAbs) are depicted. (B) A graph presenting results of an actual experiment out of more than three with similar results is shown. Experiments were performed in duplicate (data from ∼10 000 single-cell events). Bars, mean values (MFI). (*) P < 0.05; (**) P < 0.05 ANOVA with Bonferroni post-hoc test.

Cy3-tagged 22-bp oligonucleotides are transferred from BL2 cells to HVS CD4⁺ T cells. Dot plots of FACS analysis depict total live-cell gate records of HVS T cells and donor BL2 cells transfected with 22bpCy3 alone and after coculturing (see the Materials and Methods for details). (A) HVS T cells alone are positive for CD3 and negative for Cy3. (B) BL-22bpCy3 transfectants alone are positive for Cy3 and negative for CD3. (C,D) HVS T cells and BL2-22bpCy3 were cocultured for 90 min either at 37°C (C) or at 4°C (D) and then labeled for CD3 and analyzed by FACS. Appearance of CD3⁺/Cy3⁺ cells indicates 22bpCy3 acquisition by HVS T cells. Numbers in the quadrants represent the percentages of cells within each quadrant. Results shown are of a typical experiment performed in duplicate (data collected from ∼10,000 single-cell events). Similar results were obtained in >10 experiments.

T cells acquire nonautonomous miR-127 and EBV-viral miRNAs from B cells. (A) Dot plots demonstrating the isolation of highly purified human CD3⁺ T cells composed of >99.5% singlet cell from cocultures of CD3⁺ T cells and BL2- pre-miR-127/22bpCy3 cotransfectants using stringent FACS gating strategy are shown. The 22bpCy3 served as a reporter for small RNA transfer. Side scatter area (SSC-A)/forward scatter area characteristics (FSC-A) plot (panel i) and the FSC-H (height)/FSC-W (width) plot (panel ii) are shown where the arrow denotes the singlet CD3⁺ T cells consisting of two populations of low and high Cy3 content (panel iii) that were sorted into CD3⁺ cells Cy3^low (panel iv) and Cy3^high (panel v). (B) Levels of miR-127 in the sorted CD3⁺ Cy3^high and CD3⁺ Cy3^low cells and in CD3 Cy3-negative cells (from T cells that were not cocultured) as determined by real-time PCR (qPCR). Data are expressed in terms of fold increase in miR-127 content (means, n = 2) and represent a typical experiment out of three with similar results.

The vast majority of oligonucleotides are transferred from BL2 to T cells in a contact-dependent manner. (A) Overlaid dot plots of FACS analysis depict the populations of HVS T and donor BL2-22bpCy3 cells recorded after 1, 5, and 24 h of coculturing (red dots) or after similar coculturing in Transwell (0.4-μm-pore membrane) in which the two cell types are physically separated from each other (green dotes, only the HVS cells are shown). Cy3-positive/CD3⁺ HVS T cells (acquired 22bpCy3) are apparent already after 1 h of coculturing and only after 5 and 24 h of Transwell coculturing. (B) The mean fluorescence intensity (log MFI) of 22bpCy3 recorded in CD3⁺ HVS T cells and in the BL2-22bpCy3 donor cells under the coculture conditions described in A as a function of coculture time. The Cy3 log MFI of HVS T cells that were cultured alone is shown as well. Results of a typical experiment performed in duplicate (data from ∼10, 000 single-cell events) are shown. Similar results were obtained in two additional experiments. (C,D) The rate of formation and the quality of the cell contact play a role in the efficiency of 22bpCy3 transfer. 2E2 T or Jurkat cells were cocultured with 22bpCy3 transfer or nontransfected Mel-526 cells for either 0.5 h or 1.5 h under various conditions as diagrammed in C. The efficiency by which Mel-526 transfectants can transfer 22bpCy3 molecules to 2E2 or to Jurkat T cells were measured by FACS as described in Supplemental Figure 5. (C) Schematic representation of the experiments and the results. (D) The actual results of the experiment, histograms represent the Cy3 intensities in the acceptor T cells measured under the different conditions. Results shown are from a typical experiment out of 3 performed in duplicate (data collected from ∼10 000 single-cell events).