Abstract
B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction.
Highlights
Playing a major role in the humoral immune response B lymphocytes are responsible for antibody production, perform the role of antigen-presenting cells (APCs) and eventually mature into memory B lymphocytes after activation via antigen binding
Recently we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) envelope proteins hemagglutinin (H), responsible for receptor recognition, and fusion
Lentiviral vectors can transduce many types of nonproliferating cells, primary human lymphocytes normally require stimulation and entry from G0 into G1b phase of the cell cycle to become transduced by these vectors [2,3,4]
Summary
Playing a major role in the humoral immune response B lymphocytes are responsible for antibody production, perform the role of antigen-presenting cells (APCs) and eventually mature into memory B lymphocytes after activation via antigen binding These properties make B lymphocytes important target cells for immunotherapy approaches [1] and to investigate basic questions in B lymphocyte related immunology. Lentiviral vectors can transduce many types of non-proliferating cells, primary human lymphocytes normally require stimulation with cytokines or other factors and entry from G0 into G1b phase of cell cycle to become transduced by these vectors. This holds true for lentiviral vectors pseudotyped with the glycoprotein of the vesicular stomatitis virus (VSVG), which are basically the ‘‘gold standard’’ to which all other lentiviral pseudotypes are compared. Using different lentiviral pseudotypes it was observed that in resting lymphocytes post-entry steps like completion of reverse transcription, nuclear import and chromosomal integration of the transgene do not occur [2,3,4]
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