1. What is the extent of human immune-reconstitution in the different humanized mice models?
In our experience with the three humanized mouse models using multiple donor tissues we observe a modest degree of animal-to-animal variability in the levels of engraftment. Engraftment is measured by the total percentage of human CD45 (pan-lymphocyte marker) cells in the total PBMC isolated from the mice. 60-80% of all mice show 30-80% human CD45 cells between 3 and 5 months, with mean engraftment levels higher in hNSG and GTL mice compared to the BLT model. In 5% of hNSG and GTL mice engraftment as high as >90% have been observed. The human B- and T-cell development profile of the BLT and GTL mice are comparable while T-cell development is lagged in hNSG.
Figure 1. Human immune-reconstitution in the BLT mouse model. (A-D) Flow cytometry analysis of the total human hematopoietic cells (hCD45+) along with human B (CD19+) and T (CD3+) lymphocytes in the peripheral blood (PB), spleen (Sp), bone marrow (BM) and the thy-liv implant (Thy) of a representative BLT mouse 16 wk post-engraftment. Numbers indicate percentages of the total gated (indicated above each plot) cell populations. Percentages of endogenous mouse hematopoietic cells (mCD45+) are also shown in the different tissues of the BLT mice. Except for anti-mCD45, all antibodies were specific for the indicated human cell surface markers (Biswas et al, 2011).
2. Do these mice develop characteristic lymphoid follicles in secondary lymphoid tissues?
Histology and immunostaining have been performed with the spleen tissues from reconstituted hNSG and GTL mice. We observe distinct lymphoid structures including the presence of distinct B and T cells zones in a well defined lymphoid follicle. The staining pattern is comparable to that observed in normal human lymphoid tissues.
3. Is the reconstructed human immune system functional in these mice?
We have primarily analyzed the humoral immune response in the humanized mice. Groups of immune-reconstituted BLT mice 4 months after transplantation were immunized with a combination of two recombinant viral antigens, HIVgp140 and WNV envelope (10 ug each) in combination with IC31 adjuvant. Mice received immunizations on D0, 21 and 45 and sera/plasma following each immunization was analyzed by ELISA to detect antigen specific human IgM and IgG. As shown in Figure 2, a single immunization generated significant levels (over preimmune D0 sera, p<0.0001) of specific IgM and IgG responses. However, the specific IgG response was >5 fold lower than IgM response.
Figure 2. Antigen specific antibody responses in immunized BLT mice. (a) HIVgp140 (left) and WNV-E (right) specific human IgM response as detected by ELISA in BLT plasma samples obtained on day 0 prior to immunizations (D0) and on day 15 following the first immunization with recombinant viral antigens (D15). (b) HIVgp140 (left) and WNV-E (right) specific human IgG responses in D0 and D15 BLT plasma samples as detected by ELISA. Each symbol is representative of a single BLT mouse. ** P < 0.0001 determined by two-tailed Mann-Whitney U test to analyze significant differences between median values of the data sets (D0 vs. D15). Individual plasma samples were tested at a 1:100 dilution (Biswas et al, 2011).