Figure 6.

CART33 and CART123 treatment results in similar hematopoietic toxicity in two different humanized xenografts models (representative of six independent experiments). (a) Schema of Model no. 1: humanized immune system (HIS) mice were bled retro-orbitally 6–8 weeks after injection of human CD34+ cells derived from the fetal liver to confirm engraftment of human cells. Mice were then treated with either CART33, CART123, UTD cells (1 ×10⁶ cells each) or with no treatment and followed by serial weekly retro-orbital bleedings. Mice were then euthanized on day 28, and organs were harvested and analyzed. (b) CART33 or CART123 treatment results in significant reduction in myeloid progenitors (CD34+CD38+) and in hematopoietic stem cells (CD34+CD38 –). Representative FACS plots and summary statistics from bone marrow analysis by flow cytometry on day 28 at the conclusion of the experiment, gated on singlets, huCD45^dim, Lineage negative. (c) CART33 treatment results in reduction of the CD34+ compartment in the bone marrow by immunohistochemistry. Sections of the femur were taken from the mice on day 28 after treatment with UTD cells or CART33 cells. huCD45 and CD34 staining by immunohistochemistry was performed. No difference in huCD45 between control T cells and CART33, although both these groups show less huCD45 staining likely consistent with an allogeneic human-anti-human effect. There was specific reduction of CD34+ cells in mice treated with CART33. (d) Schema of Model no. 2: NSGS mice received busulfan intraperitoneally followed by 2 × 10⁶ T-cell depleted bone marrow cells from a normal donor the following day. Engraftment was confirmed by flow cytometric analysis of peripheral blood after 4 weeks, and mice were then treated with 1 × 10⁶ autologous CART33, autologous CART123 or UTD cells. Mice were then followed with retro-orbital bleeding on days 7 and 14 and were euthanized for necropsy on day 21. (e) CART33 or CART123 treatment results in significant reduction in myeloid progenitors (CD34+CD38+) and in hematopoietic stem cells (CD34+CD38 –). Representative plot of bone marrow analysis by flow cytometry on day 21. CART33 and CART123 treatment resulted in significant reduction in myeloid progenitors (CD34+CD38+) and in hematopoietic stem cells (CD34+CD38 –), Gated on huCD45^dim, Lin – . (f) Summary statistics from peripheral blood analysis by flow cytometry on day 14.