Figure 1.
HIS mouse T cells express the IL-7Rα and exhibit increased viability in response to hIL-7 in vitro.
a. Human immune cell populations were analyzed in lymphoid tissues from 20 week old HIS mice by flow cytometry to determine the extent of T and B cell engraftment. b. Peripheral blood from HIS mice or normal human donors was stained with either fluorophore-conjugated isotype control (clear histogram) or anti-hIL-7Ra antibody (grey histograms) and analyzed by flow cytometry. c. HIS mouse splenocytes (1×106) or lymph nodes cells (5×105) were cultured in increasing amounts of hIL-7 for 7 days. Cells were stained with antibodies against CD3, CD4 or CD8 as well as 7-AAD and analyzed by flow cytometry to quantify the number of live cells of each subtype.
Figure 2.
Delivery of hIL-7 to Rag2-/-γc-/- using a lentiviral vector.
Schematic of the lentiviral vector used to deliver hIL-7 or luciferase. b. Expression of luciferase was assayed using Xenogen imaging two months after intravenous injection of Rag2-/-γc-/- mice with 2×108 infectious units of luciferase expressing lentiviral vector. Localized expression from spleen (SP), bone marrow (BM), and liver (LV) are illustrated. c. Expression of hIL-7 was assayed for six months in the serum of Rag2-/-γc-/- mice following a single intravenous injection of either 1×108 or 4×108 infectious units of IL-7 expressing lentiviral vectors. Four mice were used per group, and the average and SEM are shown.
Figure 3.
Lentiviral vector delivery of hIL-7 promotes homeostatic proliferation of adoptively transferred human T cells in Rag2-/-γc-/- mice.
a. Serum concentrations of hIL-7 detected by ELISA three weeks after intravenous administration of 9×107 or 1.7×108 IU of lentivirus expressing either luciferase or hIL-7. b. The percentage of CD3+, CD4+ or CD8+ T cells of live splenocytes following one week post transfer of 2×107 CFSE labeled human PBMCs into Rag2-/-γc-/- mice from A. c. Average mean fluorescence intensity (MFI) of CFSE measured by flow cytometry in T-cell subsets quantified in B. Four mice were used per group, and the average and SEM are shown. d. Representative histograms showing CFSE loss by CD3+, CD4+ or CD8+ adoptively transferred T cells from mice receiving the control vector, low dose hIL-7 or high dose hIL-7.
Figure 4.
Lentiviral vector delivery of hIL-7 to HIS mice improves T cell levels in the peripheral blood.
a. Total human CD45+ cell engraftment in peripheral blood of cohort of mice used in this experiment prior to separation into treatment groups. b. Serum concentrations of hIL-7 detected by ELISA at 18 weeks of age in mice receiving intravenous low (1×108 IU) or high (5×108 IU) dose lentivirus expressing either luciferase or hIL-7. c. HIS mice were injected with 1×108 (low dose) or 5×108 (high dose) hIL-7 or luciferase expressing lentiviral vectors at 8 weeks of age (indicated by black arrow). The percentages of CD3+ and CD19+ peripheral blood cells (as a percentage of total human CD45+ cells) were determined from 8 to 18 weeks of age by flow cytometry. 5–7 mice were used per group, and the average and SEM are shown.d. Proportion of CD3 and CD19 expressing cells in peripheral blood of a representative luciferase or hIL-7 expressing HIS mice at 18 weeks of age as compared to a normal human donor. e. For each mouse group, human cell engraftment was determined by calculating the percentage of human CD45+ cells of total CD45+ cells. The percentage of CD3+ CD4+ and CD3+CD8+ T cells in the peripheral blood was also determined. CD3+ naïve (CD27+CD45RA+), effector (CD27+CD45RA-) and memory (CD27-CD45RA-) phenotype T cells were quantified by flow cytometry.
Figure 5.
Lentiviral vector delivery of hIL-7 improves T cell levels in the spleens and lymph nodes of HIS mice, and increases BCL2 expression.
Spleens were removed from hIL-7 (low dose group) or luciferase expressing mice and weighed. b. Splenic sections were H&E stained (scale bar = 1 mm). c. Spleens and lymph nodes were processed into single cell suspensions and flow cytometry was used to analyze the percentage of human CD3+ versus CD19+ cells in the different groups. CD3+ cells were analyzed to quantify CD4+ and CD8+ subsets. d. Absolute cell numbers for the indicated lineages were determined using splenocytes from hIL-7 expressing or control mice. e. Serial splenic sections were stained with antibodies against human CD3 (scale bar = 100 µm) or CD20 (scale bar = 100 µm), and another set with CD3 (scale bar = 100 µm) and BCL2 (scale bar = 100 µm). f. Serum from both low and high dose hIL-7 expressing mice or luciferase controls was assayed to determine the concentrations of total IgM or total IgG.
Figure 6.
Higher CD3+ Splenic T cell numbers in HIV infected HIS mice expressing hIL-7.
IL-7 or luciferase expressing HIS mice were infected with the HIV strain JR-CSF and peripheral blood was assayed by flow cytometry to determine the percentage of CD4+ of CD3+ human T cells. b. The HIV genome copy number in the peripheral blood plasma from the two groups of mice was quantified following 6 weeks of infection. c. The human CD45+, CD3+, CD4+, CD8+ and CD19+ splenocyte numbers were quantified by FACS 6 weeks after infection by HIV. d. Fixed Spleen sections from HIV infected luc and hIL-7 mice were stained for p24 (left, scale bar = 100 µm). The number of p24 positive cells in a given area (1 mm2) of a lymphoid follicle was determined (right). Data represent 8–9 mice per group, and the average and SEM are shown.