Fig 1.
Four strain comparison of engraftment.
A) Total human engraftment (CD45+) in the BM of mice at 8 and 16 weeks. B) Lineage composition of the human grafts from the BM, PB and spleen. B cells (CD19+), myeloid cells (CD13+ and CD33+), and T cells (CD3+) were assessed within the CD45+ population. C) PB CD45+ levels in mice engrafted with 6 unique UCB CD34+ preparations 6–8 weeks after engraftment. D) PB grafts from mice in “C” were plotted against the BM grafts from the same mice. The slopes of the trendlines were judged to be different by linear regression analysis, Prism 7 software, p = 0.0023. E) Lineage analysis of BM and PB for the grafts in “C”. F) Wright Giemsa stained cytospins of FACS sorted CD45+ cells from BM and spleen. G) Human cytokine serum concentrations in humanized mice. H) Flow cytometric analysis of BM grafts at 16 weeks from some mice included in experiments presented in panels A and B. I) Average total BM WBCs in 1 femur and 1 tibia (combined) were determined for NSG and NSGS mice at 15 weeks of engraftment. Total cellularity was not significantly different. Asterisks indicate p value less than 0.05 by Student’s T Test.
Fig 2.
Differential myeloid and NK differentiation in NSGS compared to NSG.
A) Representative FACS analysis of BM from UCB CD34+ engrafted mice co-stained with CD11b and CD16 antibodies at 15 weeks of engraftment. Average and standard deviation of a cohort of mice stained and analyzed as in A and represented as percentage of B) CD11b+ cells or C) CD45+ cells. D) Average percentage of CD56+ NK cells in BM of the same engrafted mice. Asterisks indicate p value less than 0.05 by Student’s T Test.
Fig 3.
Reduced platelets and lack of RBCs in NSGS PB.
A) PB from UCB-engrafted mice was stained for platelet markers CD41 and CD61. Shown are average and standard deviation. B) Representative FACS plots. C) Whole blood staining demonstrating a lack of human CD235a RBCs in both NSG and NSGS PB. Mice were engrafted for 15 weeks at the time of data collection. Asterisks indicate p value less than 0.05 by Student’s T Test.
Fig 4.
Reduced CD34+ cells in BM are myeloid skewed.
A) Averages and B) representative FACS analysis of UCB-engrafted BM demonstrating higher CD33+ and lower CD19+ co-staining on CD34+ cells from NSGS mice engrafted for 15 weeks. C) Primitive CD34+CD38- cells were also significantly reduced in NSGS BM (flow example also shown in B). Asterisks indicate p value less than 0.05 by Student’s T Test.
Fig 5.
NSGS have increased sensitivity to SRCs.
A) Whole BM from primary engrafted NSG and NSGS mice (15 weeks) was used for secondary transplant into NSGS mice. Only NSG BM had detectable SRC activity at 8 weeks after transplant. B) BM from primary NSG mice (15 weeks) was used for paired secondary transplant into NSG and NSGS mice. The NSGS mice engrafted significantly better at 8 weeks. C) A series of UCB CD34+ preparations were engrafted into non-conditioned NSG and NSGS and BM engraftment was measured at 10 weeks. Asterisks indicate p value less than 0.05 by Student’s T Tests.
Fig 6.
Rapid T cell differentiation in NSGS mice.
Representative flow plots from week 16 engrafted mice showing lineage distribution in A) spleen and B) PB. C) Flow plots showing potential T cell contamination in CD34+ isolations that was removed by FACS sorting CD34+CD3- cells. D) Kinetics of total human, B, myeloid, and T cells engraftment in the PB of NSG and NSGS mice engrafted with the same FACS sorted UCB CD34+CD3- samples. E) Thymus FACS analysis shows normal double positive primitive T cells. F) PB CD4 and CD8 frequencies among CD3+ T cells. G) Spleen Tregs were enumerated. A representative flow plot example is shown in H and the data is displayed as percentage of CD4 cells in I. J) RA/RO ratios in spleen CD3+ cells. Panels E-J were completed 15 weeks after engraftment. Asterisks indicate p value less than 0.05 by Student’s T Test.
Fig 7.
Mature T cells expand similarly in NSG and NSGS as shown by GVHD models.
A) Individual mouse weights over time after tail vein injection of adult PB MNCs. NSG (blue lines) and NSGS (red lines) experience weight loss with similar kinetics. Conditioned mice (filled boxes) show weight loss before non-conditioned mice (open boxes). B-C) Survival of the mice in panel A. D) Levels of CD3+ T cells in mice at 2 weeks. E) Levels of CD4 and CD8 cells at 2 weeks. F) Ki-67 staining reveals no changes in the cycling of either CD4 or CD8 T cells between strains. G) Representative plot used to generate panel F.
Fig 8.
Humanized NSGS mice exhibit proper splenic organization.
H&E or the indicated antibody stains were performed on formalin fixed spleen tissue from long term engrafted mice (16 weeks).
Fig 9.
Humanized NSGS mice have more functional immune systems than humanized NSG mice.
A) FACS analysis showing the average population distribution for the indicated marker combinations at 15 weeks of engraftment. B) Representative flow plots. C) IgD/IgM/IgG surface expression was measured on B cells from spleens at 15 weeks. D) Serum from long term engrafted mice (15–16 weeks) was tested for the presence of human IgG and IgM. E) Mice immunized three times with a toxoplasma extract were challenged with a final injection of extract or PBS into the footpads. Swelling was measured 24 hours later and thickness of the PBS-injected footpad was subtracted from the thickness of the extract-injected footpad. F) Splenic cell isolates from these mice were subjected to STAg extract or PBS in vitro and the media was tested for release of IFNy. G) Serum from the mice was tested for toxoplasma specific IgG and IgM antibodies by ELISA assays. Asterisks indicate p value less than 0.05 by Student’s T Test or paired T Test (panel D, F).