br Materials and methods br Results br Discussion Using a
Materials and methods
Discussion Using a dual-promoter lentivirus-transduced BM-HSC transplantation model, we report here that both transgenes (i.e. murine invariant chain (Ii) and the GFP reporter) can be detected in various APC cell types from different organs (Fig. 1). Thus, this lentiviral vector using two ubiquitous promoters – the housekeeping Elongation Factor 1a (EF1a) for Ii and Murine Stem Cell Virus (MSCV) for GFP – is efficient in expressing the two transgenes together. However, imbalanced expression of Ii and GFP was observed, with higher expression of GFP than Ii in general and with correlation of GFP and Ii only in monocytes/macrophages (Figs. 1 and 2). This imbalance may be due to vector rearrangements and/or deletions, epigenetic modification(s) or competition between promoters or enhancers for factors or competition for ribosomal association (Curtin et al., 2008). Although the MSCV promoter is functional in human HSC (Choi et al., 2001) and has been optimized in murine embryonic stem cells with additional CPG mutations at LTR region (Swindle et al., 2004), we found that it may be specifically inhibited in the murine HSC environment both in vivo and in vitro (Wang et al., 2013). On the other hand, EF1a induced the highest transgene expression in human HSC (in vitro) and their progeny cells (in vivo) among tested promoters, including MSCV (Sirven et al., 2001; Woods et al., 2002). Even with the better performing promoter EF1a to drive Ii expression, we still observed lower levels of Ii than of GFP encoded by the commercially optimized codons (Figs. 1 and 2). Thus, further improvement of expression of the gene of interest by optimization of the vector and/or gene codons (Ellis, 2005; Moreno-Carranza et al., 2009) or by using a cell-specific promoter (Cui et al., 2002) likely is warranted. As we only observed the correlation between GFP and Ii in monocytes/macrophages and there are technical difficulties detecting the GFP+Ii+ population (i.e. Ii detection requires permeabilization, whereas this procedure bleaches the fluorescence of GFP), the use of GFP as a pan-representative for Ii should be done with caution in this setting. We also observed the highest levels of Ii and GFP expression in macrophages, intermediate levels in DC, and lowest expression in TAK-875 from blood, spleen and PLN (Fig. 1). The trend is not observed in BM. The higher level of transgene expression in myeloid compared to lymphoid compartments has been reported (Amsellem et al., 2002; Mostoslavsky et al., 2005). The same trend was observed in progeny derived from HSC transduced with oncoretrovirus (Klug et al., 2000). Possible mechanisms underlying lower transgene expression in B cells include chromatin remodeling accompanying B cell commitment, the absence of positively acting transcription factors or the presence of silencing factors, and/or higher levels of DNMT3 activity in lymphoid cells compared to myeloid cells (Klug et al., 2000). These factors all contribute to silencing of transgene expression at the transcriptional level. It also is possible that the EF1a promoter works better in myeloid than in lymphoid compartments. As DC include both myeloid and lymphoid subsets (Martin et al., 2000), the transgene level is expected to be intermediate among the three cell types. Blood and BM are the most common samples collected for detecting transgene expression in mice, nonhuman primates and humans after administration of HSC-mediated gene therapy. This is useful for some therapeutic transgenes carried by blood cells (Aiuti et al., 2013; Biffi et al., 2013; Cartier et al., 2009; Kim et al., 2009; Sirven et al., 2001; Trobridge et al., 2008). However, the expression of therapeutic genes can be substantially lower in target organs, such as the lung, compared to blood (Aguilar et al., 2009). In addition, the expression of certain immune molecules, like MHC class I molecules, derived from lentivirus-transduced HSC were only transiently detected cells at the protein level in peripheral blood, although their mRNAs were consistently expressed (Zhang et al., 2005). Lower levels of GFP driven by EF1a or an APC-specific promoter were observed in spleen compared to BM (Cui et al., 2002). Here, we did not observe any correlation between Ii expression levels in BM and PLN in B cells, myeloid cells and DC. However, a confounding variable is that the markers we used allow inclusion of cells at different development stages. For example, B220+ cells in BM contain 4 stages of premature subsets before mature B cells (Nagasawa, 2006), whereas B220+ cells in PLN and spleen are mostly mature B cells. This issue needs to be considered, especially when analyzing BM. Blood Ii level correlated with PLN Ii level only in DC (Figs. 3 and 4). Thus, blood can be used as an indicator of Ii transgene expression in certain, but not all, cell compartments.