Analysis of ROR1 Protein Expression in Mice with Reconstituted Human Immune System Components

Figure 5

Engraftment of TCL-1-transduced human CD34⁺ cells in NSG mice could induce ROR1-expressing tumors. (a) Human CD34⁺ cells isolated from fetal liver tissues were transduced with lentivirus expressing TCL-1 GFP. Frequency of GFP-positive CD34⁺ cells after 4 days of lentiviral transduction is shown. (b) CD34⁺ cells transduced with lentivirus expressing TCL-1 (TCL-1 transduced) and control lentivirus (Ctr transduced) or untransduced were harvested and lysed 4 days after infection. Protein lysates were separated by gel electrophoresis, transferred to PVDF membranes by Western blotting, and probed with TCL-1-specific antibody. The blot was also probed for GAPDH as a loading control. (c) Peripheral blood from NSG mice transplanted with lentiviral-transduced CD34⁺ cells was analyzed 3 months after reconstitution. The frequencies of different immune cell compartments are indicated. Frequencies of human CD45⁺ cells within the leukocyte gate, frequencies of GFP⁺ cells, CD19⁺ B cells, NKp46⁺ NK cells, and CD3⁺ T cells within human CD45⁺ cells, and frequencies of CD4⁺ and CD8⁺ T cells within CD3⁺ cells are shown (left). The composition of the GFP⁺ cells is shown (middle), and the frequency of CD19⁺ and ROR1⁺ cells within human CD45⁺ cells in the blood, bone marrow, and spleen of the mice is presented (right). Horizontal lines represent mean and SD. Data are from 2 different reconstitution cohorts with CD34⁺ cells derived from the same donor. (d) The mouse with the highest GFP⁺ cells in the blood developed a tumor-like lump at the back as pointed by the arrow (left). Cells isolated from the tumor-like lump were analyzed by flow cytometry for the expression of human CD45 and GFP (left). The gated human CD45⁺ cells were further analyzed for CD19, ROR1, CD5, and CD23 expression. The ROR1⁺CD19⁺ cell population is represented in blue.

(a)

(b)

(c)

(d)