A Xenograft Model of Lymphocyte-Independent, Effector-Driven Macrophage Activation SyndRome Amenable to Anti-CD33 and Anti-IL-6R Treatment
Authors: Wunderlich Mark , Stockman Courtney , Devarajan Mahima , Ravishankar Navin , Sexton Christina , Kumar Ashish R , Mizukawa Benjamin , Mulloy James C. 
Published: November 2019

Abstract
Macrophage Activation Syndrome (MAS) is a form of secondary hemophagocytic lymphohistiocytosis (HLH) that frequently complicates various rheumatoid disorders. Similar to HLH, these disorders present with chronic inflammation and immune dysfunction. Most typically, an identifiable infection triggers an immune response that is either ineffective due to genetic inactivating mutations that affect lymphocyte cytotoxicity, as in primary HLH, or uncontrolled and characterized by expansion of activated T and macrophage cells as in the case of MAS. While lymphocyte function is generally thought to be a crucial aspect of the disease state, abnormal myeloid cell activation by genetic activation of inflammasomes or repeated TLR stimulation was recently shown to result in MAS. These studies have implicated several cytokines, including SCF, IL-3, and GM-CSF in the disease process. A lymphocyte-independent model could be useful to isolate the myeloid contribution to MAS and would allow the testing of additional therapeutic strategies that target the effector cells directly. Such novel therapies are needed to address refractory cases that often prove fatal.
Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross species activities of 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 non-transgenontrols. After engraftment with UCB, these mice develop severe, fatal MAS characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias and evidence of chronic inflammation. The BM becomes strikingly hypocellular and spleens are significantly enlarged with evidence of extramedullary hematopoiesis. Activated macrophages engaged in hemophagocytosis are readily detectable in the spleen and liver. Mice also show elevated body temperature and sCD25, indicating the presence of activated lymphocytes.
Treatment of mice with the CD52 inhibitor alemtuzumab eliminated the human graft and resolved MAS, implicating a human cell and demonstrating the reversibility of the phenotype. However, there was no response to lymphocyte suppressive therapies such as steroids, intravenous 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, demonstrating the utility of targeting the effector macrophage cells directly in this model.
Using a multiplex ELISA assay, TNF alpha and IFN gamma were low to absent in affected mice, however, IL-6, IL-10, MIP-1 alpha, and MIP-1 beta were consistently upregulated. IL-6, MIP-1 alpha, and MIP-1 beta were lost after gemtuzumab ozogamicin therapy, but not after combined B/T cell ablation with rituximab and OKT-3. Interestingly, IL-10 and sCD25 was suppressed by either treatment. Since the IL-6R antibody tocilizumab is currently being evaluated in a clinical trial for HLH, we examined the efficacy of tocilizumab injections in our model and found delayed progression of anemia and a prolonged lifespan for affected mice. This novel model of MAS provides a new tool for the investigation of the mechanisms driving MAS and for the testing of myeloid-directed therapies in a human cell model.

Disclosures
No relevant conflicts of interest to declare.