IFN-γ Inhibitors and Ruxolitinib Therapy for Acquired Severe Aplastic Anemia in an Ex-Vivo Model

Background:

Severe aplastic anemia (SAA) is a life-threatening disorder that is associated with multiple etiologies, both inherited and acquired. In acquired SAA, oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Currently, the first line treatment for patients who have a suitable HLA matched donor is a hematopoietic progenitor cell transplant (HPCT). When HPCT is not possible, due to lack of a closely matched HLA donor and/or concomitant co-morbidities, then the treatment of choice is immunosuppression with anti-thymocyte globulin, cyclosporine and eltrombopag (ELT)(Georges et al, Blood, 2018). Alvarado et al (Blood, 2019) recently demonstrated that ELT bypasses the inhibitory effect of IFN-γ by alternatively activating TPO signaling. However, ELT cannot overcome other IFN-γ mediated effect through JAK-STAT1 phosphorylation or apoptosis via Fas/FasL. Alternative therapies are in great need for patients with aSAA as treatment response is sub-optimal.

Objective:

To determine the effects of IFN-γ neutralizing antibodies or Ruxolitinib on HSPCs survival, proliferation and differentiation in an ex vivo culture of human CD34+ cells in the presence of IFN-γ and TNF-α.

Design/Methods:

Human CD34+ HSPCs were isolated from cord blood, based on CD34 microbeads magnetic selection (Miltenyl Biotec, Germany). The CD34+ cells were cultured in StemSpan Serum-Free Medium II (STEMCELL Technologies) supplemented with 5 ng/mL human stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and 5 ng/mL recombinant human TPO. A 1x10 ⁵ CD34+ HSPCs were seeded in a 96 well plate. HSPC alone, with and without IFN-γ (100 ng/mL) and TNF-α (10 ng/mL), were the negative and positive control, respectively. Specific IFN-γ neutralizing antibody, B27 (BD Pharmingen), MD1 (BioLegend) and B133.5 (ImmunoTools) or Ruxolitinib (Jakafi, Incyte) were added to the culture and HSPC were harvested and assayed for their survival at day 7 and 14. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO ₂. Also, we assessed the multi-lineage differentiation capacity with a selective colony forming units (CFU) assay. Fourteen days after co-culture of each experimental treatment, 500 CD34+ cells were seeded in 6-well plates Smart Dish™ (STEMCELL Technologies). Big burst forming units of erythroid (BFU-E), CFU of granulocyte and megakaryocyte (GM) and granulocyte, erythrocyte, macrophage, megakaryocyte (GEMM) were counted and compared between the experimental groups. The signaling pathways were determined using phospho flow-cytometric analysis of pSTAT1, pSTAT3, pSTAT5. All statistically analyzed data is represented as mean ± SD. Differences between groups were analyzed by multiple 2-tailed unpaired Student t tests using Excel. Statistically significant differences were represented as *P <0.05, ** P <0.01 and *** P<0.001.

Results:

The number of CD34+ cells at day 7 and day 14 of culture in the presence of IFN-γ and TNF-α was significantly lower (21% ± 3 (p<0.0006) and 15% ± 6 (p<0.02), respectively, than that of the control (Fig 1). Importantly, the myelosuppressive effect of IFN-γ and TNF-α was significantly rescued by the addition of Ruxolitinib (at day 14, p<0.05) or IFN-γ neutralizing antibodies (B27 and B133.5, respectively) (day 7 and 14, p<0.01). Improvement in HSPC survival ranged between 1.5-3.3 fold compared to our negative control. Based on the CFU analysis, the CD34+ cells cultured in the presence of B27, B133.5 or Ruxolitinib, were able to produce more CFU at day 7 (p<0.01, p<0.01 and p<0.05 respectively)(Fig 2A). Additionally, Both B27 (p < 0.01) and Ruxolitinb (p ≤ 0.001) were found to produce more CFU-GM on day 14 (Fig 2B). Phospho flow-cytometry demonstrated a significant decrease in STAT1 phosphorylation of CD34+ cells in the presence of B27 and B133.5 (p<0.05, p<0.001, respectively).

Conclusions:

Our preliminary studies supports the potential benefits of utilizing IFN-γ neutralizing antibodies or Ruxolitinib to improve HSPC survival, proliferation and differentiation in aSAA. Future studies will need to be done to investigate the exact mechanisms of action and the effects of IFN-γ neutralizing antibodies in an animal model of aSAA.

Figure 1

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Figure 1

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