We report here the generation of a novel in vivo system enabling the somatic and flexible genetic manipulation of Blimp-1-producing cells through ectopic expression of TVB, an ALV receptor, under the control of transcriptional elements belonging to the gene encoding Blimp-1, Prdm1
. We created this system because perturbation of the growth and/or homeostasis of Prdm1
-expressing hematopoietic cells has been directly linked with disease processes, such as tumors of the B-cell lineage and autoimmunity (29
). Moreover, expression of Prdm1
has been shown to play a central role in the lineage commitment of specific embryonic and adult lineages, some with stem cell properties. An animal system that enables the flexible genetic manipulation of Prdm1
-expressing cells could be a valuable tool for investigating these diverse physiologic and pathological processes in vivo.
To achieve cell-specific transduction of lineages regulated by Blimp-1, we ectopically expressed the ALV receptor TVB, driven by regulatory elements belonging to the Prdm1
locus. This is the first transgenic system employing the ALV receptor TVB; among the various ALV receptors, only the receptor TVA has previously been utilized for tissue-specific gene delivery in mammalian systems (13
). TVB does not share structural similarity with TVA and is encoded by a separate locus in the chick genome. The expansion of the spectrum of ALV receptors that can be used for generation of mammalian transgenic systems is important because it will allow lineage-directed gene targeting in the same animal following gene transduction through alternate ALV receptors. We envisage several attractive applications of this technology: it can be used to address cell-of-origin questions in cancer; it can also provide an attractive approach to probing the complex interactions between tumor cells and nonmalignant cells in the microenvironment.
To trace the expression of TVB in this system, we expressed the ALV receptor as a fusion to mRFP. The mRFP moiety of the fusion receptor was readily detectable by flow cytometry with a 568-nm laser. Using this approach, we detected TVB-mRFP expression in a small subpopulation of splenocytes and bone marrow cells in young animals undergoing immune responses to sheep red blood cells, a T-cell-dependent immunogen. Analysis of the TVB-mRFP-expressing population by surface immunophenotype in immunized animals confirmed that the majority consisted of CD138+
plasma cells and CD4+
T cells. Strikingly, we did not find a significant proportion of CD8+
T cells coexpressing TVB-mRFP. This may be due to the specific conditions employed to induce T-cell-dependent immune responses, i.e., the immunization of young animals with sheep red blood cells. We postulate that different conditions, such as viral challenge, may elicit a substantially higher proportion of CD8+
effector cells (5
). The fluctuating makeup of the mRFP+
population under diverse experimental conditions is further underscored by the finding that in nonimmunized, age-matched animals, the T-cell component of this population was relatively underrepresented (Fig. ). We also detected a reproducible B220+
population coexpressing TVB-mRFP. We postulate that at least a component of this population may include proliferative plasma cell precursors generated in the course of T-cell-dependent immune responses. These cells, in turn, may constitute the precursor of Prdm1
-expressing B-cell malignancies arising in the germinal center, such as plasma cell myeloma and some diffuse large B-cell lymphomas.
We subsequently defined conditions for efficient transduction of Prdm1
-expressing lymphocytes by ALV vectors incorporating a subgroup B envelope. To our knowledge, this is the first ALV-based system, and one of the few retroviral systems, allowing efficient retroviral transduction of in vivo-activated mature lymphocytes (21
). Because only a minority of TVB-expressing cells are in cell cycle (e.g., most CD138+
cells are quiescent plasma cells), the fraction of susceptible target cells transduced under the conditions delineated in this paper is likely to be significantly higher than the proportion (5%) of total cells transduced (Fig. ). We determined that a high MOI (20 to 40) is required for detection of reporter fluorescence in transduced primary lymphocytes but that transduction at a much lower MOI was adequate to detect reporter fluorescence in established human lymphoid cell lines engineered to stably express TVB. Furthermore, we found that the incorporation of an internal CMV promoter in the viral construct appears to allow a subset of cells to exhibit extremely high levels of reporter fluorescence; similar levels of reporter GFP fluorescence were not obtained when expression of the reporter gene was driven by the native ALV LTR. This finding may be relevant to experiments in which high levels of gene expression are desired.
We are initially using Prdm1:TVB-mRFP transgenic animals as a basis for developing a flexible animal model for multiple myeloma, an incurable cancer of plasma cells that has hitherto proven difficult to model faithfully. More generally, in the B-cell lineage, genetic manipulation of Prdm1-expressing effectors could further illuminate the molecular processes underlying commitment to a plasmacytic fate (and likely the exclusion of a memory B-cell fate). The role of Blimp-1 in T-cell maturation has only recently begun to be characterized; however, expression of this transcription factor has already been linked with differentiation, homeostasis, and/or function of memory T cells and at least a subset of regulatory T cells. The spectrum of lineages rendered susceptible to genetic manipulation in the system presented here is likely to extend to other adult and embryonic lineages regulated by Blimp-1. Thus, Prdm1:TVB-mRFP mice could prove a useful tool for studying several aspects of hematopoiesis, immunity, development, and cancer that are associated with production of Blimp-1.