Synthesis and delivery of short, noncoding RNA by B lymphocytes

Abstract

Evolutionarily conserved short (20-30 nucleotides) noncoding RNAs (microRNAs) are powerful regulators of gene expression in a variety of physiological and pathological processes. As such, means to efficiently modulate microRNA function constitute an important therapeutic opportunity. Here we demonstrate that primary B lymphocytes can be genetically programmed with nonviral plasmid DNA for the biogenesis and delivery of antisense sequences (anti-microRNA) against microRNA-150 (miR-150). Within 18 h of transfection with an anti-miR-150 construct, primary B lymphocytes secrete ∼3,000 copies of anti-miR-150 molecules per cell. Anti-miR-150 molecules released by B lymphocytes were internalized by CD8 T lymphocytes during cross-priming in vitro and in vivo, resulting in marked down-regulation of endogenous miR-150. However, such internalization was not observed in the absence of cross-priming. These results suggest that shuttling anti-miR-150 molecules from B lymphocytes to T cells requires the activation of receiver T cells via the antigen receptor. Finally, anti-miR-150 synthesized in B cells were secreted both as free and extracellular vesicle-associated fractions, but only extracellular vesicle-associated anti-miR-150 were apparently taken up by CD8 T cells. Collectively, these data indicate that primary B lymphocytes represent an efficient platform for the synthesis and delivery of short, noncoding RNA, paving the way for an approach to immunogenomic therapies.

Keywords: immunotherapy; microvesicles.

Fig. 1.

Structure of pCMV-MIR^a150 and secretion of anti-miR-150 by primary B lymphocytes transfected with pCMV-MIR^a150. (A) Schematic map of pCMV-MIR^a150 and anti-miR-150 coding sequence and transcript. (B) Intracellular anti-miR-150 detection (copy number per cell) in primary B lymphocytes transfected with pCMV-MIR^a150 harvested 18 h after transfection. (C) Secreted anti-miR-150 (copy number per cell) based on detection in culture supernatants harvested 18 h after transfection. Data points refer to single independent experiments.

Fig. 2.

Anti-miR-150 secreted by transfected primary B lymphocytes are internalized by CD8 T cells during cross-priming in vitro. (A) Scheme of experimental design of in vitro T-cell cross-priming. (B) Flow cytometry analysis showing that in vitro cross-primed CD8 T cells express CD69 and CD44 activation markers. (C) Detection of anti-miR-150 content (copy number per cell) in purified CD8 T lymphocytes cross-primed in the presence of supernatant from primary B lymphocytes transfected with pCMV-MIR^a150. CD8 T cells cross-primed without addition of transfected primary B lymphocyte supernatant or CD8 T cells from BMDC-CD8 T-cell cocultures without OVA (BMDC + OT-I CD8 + B Sup) to which the supernatant of primary B lymphocytes transfected with pCMV-MIR^a150 was added, served as controls. Dots refer to single independent experiments. (D) Fold modulation (RQ) of endogenous miR-150 in CD8 T cells of corresponding cultures.

Fig. 3.

Transfected B lymphocytes transfer anti-miR-150 to CD8 T cells during cross-priming in vivo. (A) Schematic of in vivo cross-priming. OVA (5 mg) was injected i.p. into OT-I and F5 mice. After 24 h mice were injected i.v. with 10⁶ syngeneic primary B lymphocytes transfected (<24 h) with pCMV-MIR^a150. OT-I and F5 mice were killed 48 h later, and spleen and lymph node CD8⁺ T cells were isolated and analyzed for expression of cell-surface activation markers by flow cytometry (B) as in Fig. 2. (C) Content (copy number per cell) of anti-miR-150 molecules, and (D) fold modulation of endogenous miR-150 in CD8 T cells purified from OT-I and F5 spleens after cross-priming in vivo. mir150 expression of naïve T lymphocytes from OT-I mice was set to 1.

Fig. 4.

Isolation of anti-miR-150 activity in EVs and EV visualization inside CD8 T cells after cross-priming in vitro. (A) Schematic of in vitro production and isolation of EVs from J558L cells transfected with pCMV-MIR^a150. (B) Detection of anti-miR-150 in the 120K EV-free supernatant and in the EV-rich pellet of J558L cells after short-term (96 h) transfection with pCMV-MIR^a150. Results are expressed as fold modulation and refer to the results of two independent experiments (mean ± SEM). (C) Fold modulation of anti-miR-150 inside in vitro cross-primed CD8 T cells, and appropriate controls, with or without addition of the 120K EV-free supernatant or EV-rich ultracentrifugation pellet. Results refer to two independent experiments (mean ± SEM). (D) Fluorescence microscopy analysis of CD8 T cells cross-primed in vitro in the presence of PKH67-labeled EVs (Left) or cocultured with DCs without OVA (no cross-priming) but in the presence of PKH67-labeled EVs (Right). (Magnification: Left, 20×; Right, 10×.)