A xenograft model of macrophage activation syndrome amenable to anti-CD33 and anti-IL-6R treatment

Abstract

Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross-species activities of stem cell factor (SCF), IL-3, and GM-CSF. When engrafted with human umbilical cord blood (UCB), these triple-transgenic mice produce BM and spleen grafts with much higher myeloid composition, relative to nontransgenic controls. Shortly after engraftment with UCB, these mice develop a severe, fatal macrophage activation syndrome (MAS) characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias, and evidence of chronic inflammation, including elevated human IL-6. The BM becomes strikingly hypocellular, and spleens are significantly enlarged with evidence of extramedullary hematopoiesis and activated macrophages engaged in hemophagocytosis. This manifestation of MAS does not respond to lymphocyte-suppressive therapies such as steroids, i.v. immunoglobulin, or antibody-mediated ablation of human B and T cells, demonstrating a lymphocyte-independent mechanism of action. In contrast, elimination of human myeloid cells using gemtuzumab ozogamicin (anti-CD33) completely reversed the disease. Additionally, the IL-6R antibody tocilizumab delayed progression and prolonged lifespan. This new model of MAS provides an opportunity for investigation of the mechanisms driving this disease and for the testing of directed therapies in a humanized mouse.

Figure 1. Characterization of MAS in NSGS mice.

(A) Survival of UCB-engrafted NSGS mice and nonengrafted control (CNTL) NSGS mice. (B) Serial rbc counts from NSG, NSS, and NSGS mice engrafted with the same UCB and nonengrafted controls. Average result is shown for each group. (C) Peripheral blood samples from B were analyzed for reticulocytes. (D) Persistence of CFSE-labeled rbcs in UCB-engrafted NSG and NSGS mice over time. Small-volume serial bleeds were performed, and CFSE-positive cells were enumerated by flow cytometry. Half-life was determined by solving for the equation of the line of best fit generated by Excel. (E) Serial rbc counts from anemic mice receiving transfusions. Transfusions occurred just after the baseline bleed at day 0. (F) Total BM cell counts of nonengrafted CNTL or UCB-engrafted NSGS mice. (G) Spleens from sacrificed animals in F were weighed. (H) H&E stains of formalin-fixed femurs from engrafted mice. (I) Spleen, BM, and liver samples from engrafted NSGS mice were spun onto slides and stained with Wright-Giemsa. (J) Rectal temperatures were determined for active control nonengrafted mice and UCB-engrafted hu-NRGS mice. (K) Soluble IL2-Rα (sCD25) levels in the plasma of engrafted NSG and NSGS mice were determined by ELISA. *P < 0.05 by Mann-Whitney U test. Panels F, G, and J are box and whisker plots with the bounds of the box indicating the 1st and 3rd quartiles, the line within the box showing the mean, and the whiskers comprising the range of all data points.

Figure 2. NSGS MAS is a lymphoid-independent, myeloid-driven disease.

(A) Engrafted NSGS mice with MAS were treated with weekly doses of antibodies or steroids to ablate or inhibit human B and T cell activities (IVIG, i.v. Ig; Rit, rituximab; DEX, dexamethasone). The rbc counts before and after are shown for each mouse in a representative experiment. Each treatment was repeated in additional experiments at least 3 times. (B) Newly engrafted mice were given combinations of Rit and OKT3 to prevent B and T cell reconstitution in NSGS mice. The rbc counts were determined 12 weeks after engraftment. Three mice per group were used in this representative experiment. Mean ±SD and individual mice are shown. (C) The experiment in A was repeated to include Campath and Mylotarg. Each treatment was repeated in at least 3 experiments. (D) Spleen preparations from antibody-treated mice were subjected to flow cytometry to demonstrate the degree and specificity of monoclonal antibody depletion in vivo. (E) PB rbc counts from engrafted mice or nonengrafted controls (CNTL). (F) Spleen weights and (G) BM cellularity were determined in the same mice as E. *P < 0.05 by Mann-Whitney U test. Panels E, F, and G are box and whisker plots with the bounds of the box indicating the 1st and 3rd quartiles, the line within the box showing the mean, and the whiskers comprising the range of the data points.

Figure 3. Identification and efficacy of targeting IL-6/IL-6R signaling.

(A) Levels of several human inflammatory cytokines were measured in the plasma of control nonengrafted (CNTL) or long-term UCB-engrafted NSG and NSGS mice by multiplex ELISA. n = 4 CNTL NSGS, n = 9–13 UCB-NSGS, n = 4 CNTL NSG, and n = 7 UCB-NSG samples were used for analysis. LLOQ, lower limit of quantification. *P < 0.05 by Mann-Whitney U test. (B) Plasma levels of human cytokines in UCB-engrafted NSGS mice after the indicated treatments. CAM, Campath/alemtuzumab; MT, Mylotarg/gemtuzumab ozogamicin; R/O, rituximab + OKT3. n = 11 PBS, n = 3 CAM, n = 9 MT, and n = 3 R/O. One-way ANOVA followed by Tukey HSD test was used to determine significance. (C) Tocilizumab treatment was delayed until the mice had significant loss in PB rbc counts. Treatment started after the week 17 baseline measurement. n = 17 mice per group. (D) UCB-engrafted NSGS were treated with PBS or tocilizumab (Tocil) before anemia could be detected. Serial blood counts were taken at the indicated times. Treatment started after the week 8 baseline measurement. n = 10 mice per group. Panels C and D are box and whisker plots with the bounds of the box indicating the 1st and 3rd quartiles, the line within the box showing the mean, and the whiskers comprising the range of the data points. Repeated measures 2-way ANOVA was performed on the data in C and D to determine significance. (E) Treated mice were followed long-term for survival. The log rank test was used to determine differences in survival.