Fig 2: Functional analysis of expanded murine bone marrow cells in vivo.Cohorts of irradiated Rag-1−/− mice were given 104 expanded bone marrow cells obtained from 5 FU treated wild type C57BL/6J mice after expansion with Tat-MYC/Tat-Bcl-2. A. Peripheral blood from chimaeric mice revealed the presence of mature B-cells, expressing B-cell markers B220 and CD19, (2nd panel) compared to the Rag-1−/− control (1st panel). B. Mature T-cells, expressing T-cell markers TCRß and CD4+, were also observed in the peripheral blood of Rag-1−/− chimaeric mice (2nd panel) compared to the Rag-1−/− control (1st panel). C. Lymphoid organs from the engrafted Rag-1−/− mice also stained positive for developing T- (CD4 and CD8) and B-cells (CD19xIgM) by flow cytometry. D. The expanded mouse bone marrow also differentiates into myeloid cells. Cohorts of sublethally irradiated Rag-1−/− mice (Thy 1.2) were given 104 cells derived from bone marrow cells obtained from PLJ (Thy 1.1) mice after expansion with Tat-MYC/Tat-Bcl-2. Peripheral blood from the Thy 1.1/Thy 1.2 chimaeric mice revealed the presence of Thy 1.1 myeloid cells (2nd panel, top row) compared to the Rag-1−/− control (1st panel, top row). The Thy 1.1/Thy 1.2 chimaeric mice did not stain for Thy 1.2 (2nd panel, bottom row) compared to the Rag-1−/− control (1st panel, bottom row). E. 4 weeks post engraftment the Rag1−/− Thy 1.1/Thy 1.2 chimaeric mice were euthanized and their thymii were assessed for T-cells. The thymus from the Rag1−/− Thy 1.1/Thy 1.2 chimaeric mice stained positive for TCRßxCD4 positive T-cells (bottom panel) compared to a Rag1−/− control mouse (top panel).

Fig 3: Expansion of adult human bone marrow derived cells ex vivo with Tat-MYC/Tat-Bcl-2. A. The percent CD34+ cells in culture on day 0 and day12 days of expansion B. Graphical representation of the CD34+ cells expansion from two independent adult BM samples. C. Quantification of each colony type that was observed in methylcellulose cultures seeded with 300 expanded cells from adult BM cultured in CB media supplemented with Tat-MYC/Tat-Bcl-2. Functional analysis of expanded human BM in vivo. Cohorts of irradiated NSG mice were given transplants of 1×106 cells expanded ex vivo with Tat-MYC/Tat-Bcl-2. The NSG xenochimaeric mice were euthanized 10 weeks post transplant and used to collect bone marrow cells. D. The BM cells were stained for human hCD45. E. Gating on CD45+ cells, we observed human CD34+/CD38+ cells in the BM (1st panel), CD19+ and CD3+ cells in the spleen (2nd panel) and CD11b+/CD33+ cells in the spleen (3rd panel). F. A dot plot representation of the percent CD34+ engraftment in the BM of mice transplanted of 106 adult human BM cells expanded ex vivo in CB media supplemented with Tat-MYC/Tat-Bcl-2. G. A dot plot showing the lineage+ engraftment in the spleen of mice shown of F.

Fig 4: (A) Anti-CD antibody staining for peripheral blood samples shows that the GFP(+) cells in the healthy subjects were monocytes. Nuclei were stained with DAPI. CD45 was stained using PerCP-Cy5.5-conjugated antibody. All other CD antigens were stained using PE-conjugated antibodies. (B) CD45(+)/GFP(+) cells in peripheral blood samples from the cancer patients confirmed that the monocytes showed positivity. Nuclei were stained with DAPI. CD45 was stained using PerCP-Cy5.5-conjugated antibody All other CD antigens were stained using PE-conjugated antibodies. BF, bright field. Scale bar, 10 μm.

Fig 5: Validation of RG7769 receptor occupancy (RO) assay for whole‐blood T cell RO analysis. (A) Gating strategy for RO calculation. I. In all conditions, single lymphocytes were identified by excluding cell doublets via FSC‐H versus FSC‐W followed by SSC‐H versus SSC‐W. II. Total lymphocytes were gated on via CD45 staining versus SSC‐A. III. Within the CD45+ compartment, T cells were identified via CD3 expression. A non‐T cell gate (NOT‐CD3+) was set. IV. Within total CD3+ T cells, CD4+ (Q3) and CD8+ single‐positive (Q1) T cells were identified. V. In the non‐T cell population, NK cells were identified via CD56 versus CD16 (QI‐III). Total NK cells were defined as CD56+CD16+/−. Lower right: Comparing the anti‐PGLALA‐PE staining intensity of CD3+ T cells, CD4+ and CD8+ T cells pulsed with RG7769. Orange: 1 μg/ml, red: Saturation (100 μg/ml); gray: Negative control (1000 ng/ml + mIgG2b‐isotype‐PE). (B) Dose–response of RG7769 binding to healthy donor lymphocytes. Using the gating strategy in A, CD45+ lymphocyte single cells from blood pulsed ex vivo with RG7769 (0 ng/ml➔100 ng/ml), were identified. The control group (0 ng/ml) was stained with mIgG2b‐PE isotype, cells with RG7769 with anti‐PGLALA‐PE. Five thousand concatenated CD45+ events were clustered according to CD3, CD4, CD8, CD56 and CD16 by UMAP. Left: Cell lineages identified using FlowSOM include CD4+ T cells, CD8+ T cells, CD4−CD8− unconventional T cells as well as NK cell subsets. Right: PE+ (RG7769+) T cells (blue) [Color figure can be viewed at wileyonlinelibrary.com]