AfCS Research Reports

To manipulate signaling protein expression, the AfCS plans to utilize plasmid-based shRNA- and/or siRNA-mediated RNAi or RNA antisense. These methods will require transfection or viral transduction to introduce exogenous DNA or RNA into the model cell system. Unfortunately, primary B cells are resistant to transfection by electroporation or by chemical techniques. Furthermore, transduction with Moloney-based retroviral systems has been achieved only in B cells that have been prestimulated and that are actively cycling (27, 28). However, a subset of retroviruses, the lentiviruses, can infect nondividing cells. HIV is one type of lentivirus, and recently, HIV-based lentiviral systems have been developed to transduce noncycling cells (29, 30).

To examine transduction of B cells by lentivirus, we tested two related and overlapping systems (see Methods and Protocols) for the production of nonreplicating lentivirus vectors. The data shown are for cells infected with lentivirus carrying green fluorescence protein (GFP) as a marker for successful transduction. Infection of 293T cells with unconcentrated lentivirus at a density of approximately 1 x 10⁶ virus particles/ml, using a multiplicity of infection (MOI) of 5, caused infection of >95% of cells. By concentration of the lentivirus produced using Centricon filtration units, we obtained viral titers of 10⁸ to 10⁹ infectious units/ml, as assessed by transduction of fresh 293T cells, which allowed infection of target cells at higher virus densities and MOIs. However, in multiple experiments, we detected minimal transduction (~3%) of unstimulated fresh B cells from either hbcl-2 transgenic (Fig. 18A) or wild-type mice (data not shown), even with MOIs up to 50, at densities of >3 x 10⁷/ml. Stimulation of B cells with an activation/proliferation promoting ligand, such as LPS or anti-CD40, for one day prior to infection produced a modest increase in expression of GFP relative to nonstimulated cells. In the experiment shown, approximately 6% of cells were transduced, and median fluorescence intensity of GFP in the transfected cell population increased by approximately 1.5X compared with noninfected cells (Fig. 18B). Of note, the same virus stock was successfully used to efficiently transduce the pro-B cell line Ba/F3 at >90% (Fig. 18C).

Fig. 18. Lentivirus infection of hbcl-2 transgenic B cells compared with the pro-B cell line Ba/F3. Nonstimulated and one day LPS (10 mg/ml) preactivated hbcl-2 transgenic B cells (A and B) and log phase Ba/F3 pro-B cells (C) were infected with the same batch of concentrated eGFP carrying lentivirus (made using pFUGW vector plasmid) at an MOI of 50, in the presence of 4 mg/ml polybrene. Cells were returned to culture and harvested for FACS cytometry analysis at three days postinfection. Analysis of the proportion of cells transduced and expressing GFP was performed on live cells (R1 = region 1, determined by forward and side scatter properties) for each population; R1 dot plot data is not shown for B cells (A and B) but is shown for Ba/F3 cells (C). Successful transduction was measured in terms of relative eGFP expression, compared with autofluorescence for noninfected control cell populations. Cells exposed to virus are shown in green-fill histograms, negative/mock controls shown with purple line histograms (underwent identical experimental treatments with the exception of virus addition). Representative results from multiple experiments are shown.

In other studies, success with primary T and B lymphocytes (human or mouse) has mainly been limited to infection of cells activated by cytokines and/or growth factors, suggesting that transduction of noncycling lymphocytes is inefficient (14, 15, 31-34). Lentivirus systems have been used successfully to transduce siRNA into T lymphocytes, but all published reports used T cells that were activated in vitro by mitogen or antigen and were subsequently stimulated with IL-2 (14, 15). Lentivirus vectors carrying modified promoters (human EF-1a, and improved biosafety designs) increased transduction efficiencies for some human hematopoietic cells (31), and relatively efficient transduction (>20%) of activated primary human B cells was reported with other lentivirus vector systems (32, 33). Rossi (34) recently reported higher levels of lentiviral-mediated transduction of mouse B cells stimulated with either LPS or anti-CD40, using pHR'CMV-eGFP-based lentivirus vectors. Early in our studies, on a number of occasions, we achieved levels of GFP expression comparable to those reported by Rossi, but our results were inconsistent, and this GFP expression was lost even though our virus preparations sustained transduction of control cells. When expression of GFP by B cells was observed in these experiments, it was a uniform increase in expression by all B cells, rather than by a subset, suggesting that this might represent an artifact of adsorption of GFP debris carryover from the 293T packaging cells (even though virus preparations were centrifuged and filtered prior to use; see Methods and Protocols). However, because we stopped seeing this phenomenon, and changed methods of virus production, we could not further test its origin. We conclude that B cells are, at the least, much more resistant to transduction using the current lentivirus vectors and methods than are other primary leukocytes (for which we have successfully obtained high efficiency transduction, i.e., activated primary T cells and bone marrow-derived macrophages).

In summary, we found that cultured splenic hbcl-2 transgenic B cells provided an excellent model for signaling studies in a primary mature B cell, despite being relatively refractory to gene transfection or transduction. Thus, hbcl-2 transgenic splenic B cells meet the following criteria as a useful model for the study of signaling:

ii) B cells have extended survival in culture, with high viability in the absence of stimulation/activation.

iii) B cell purity improved with culture (through loss of the ~5% of contaminating cell types).

iv) The cultured B cells provided a stable platform for experiments examining the regulation of gene expression using RNA arrays.

v) All surface phenotypes and functional responses tested were essentially maintained in cultured hbcl-2 transgenic cells for up to six days.

Despite the inability to manipulate hbcl-2 transgenic B cells for signaling studies using the current methods for RNAi, this model system does open up many exciting alternative avenues for investigation. In addition to increasing the possibilities for microarray studies of long-term gene expression changes following single or dual ligand stimulations, the possibilities with other tools of signal transduction exploration, including the use of chemical inhibitors, knock-out mice, and the measurement of functional responses following ligand stimulations, can also be greatly expanded by using the hbcl-2 transgenic background in the B cells.