Background: Immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG) and cyclosporine A is one first-line treatment option for patients (pts) with aplastic anemia (AA). One side effect of ATG is the release of interferon-gamma (IFN-γ), which is considered a major factor in the pathogenic autoimmune depletion of hematopoietic stem cells (HSC). Recently, eltrombopag (EPAG) was introduced for therapy of refractory AA pts due to its ability to improve HSC function. Clinical trials have evidenced that EPAG used alongside with IST leads to a higher response rate as compared to its later administration e.g. 2 weeks after ATG start. However, the potential underlying cause for this difference has not been clarified to date.
Aims: We hypothesize that EPAG might protect HSC from negative effects of ATG-induced IFN-γ release.
Methods: Sera of six pts with moderate to severe AA were collected pre-and post-ATG administration on days +1 and +2. Bone marrow (BM) of AA pts was collected and cryopreserved before ATG administration. In addition, we collected CD34+ cells isolated from five healthy donors (HD) undergoing HSC mobilization. HD-derived CD34+ or AA-derived BM cells were cultured in presence of pre as well as post-ATG serum from pts with AA, in combination with 5 µM EPAG and/or an IFN-γ neutralizing antibody (NAB; 2.5 µg/mL). After 48 h, colony forming unit (CFU) assay with cultured cells and intracellular staining of phospho-SMAD2/3 (known to be down-regulated in response of IFN-γ) were performed, while IFN-γ was measured via flow cytometry in collected sera.
Results: Serum IFN-γ levels were 0.56 ± 0.6 pg/mL (mean ± standard deviation) post-ATG, whereas they were 0.015 ± 0.04 pg/mL pre-ATG (n= 6; p=0.08). When we tested effects of ATG sera on the clonogenic potential (CP) of HD HSC (n=5), a significant decrease of the number of CFU colonies (NoC) treated with post-ATG (148 ± 75) sera was found compared to pre-ATG treatment (201 ± 75, p= 0.032). This negative effect was mitigated by adding EPAG to the post-ATG sera (223 ± 100; p=0.036). To specifically analyze the potential suppressive role of IFN-γ, we added a IFN-γ NAB to the post-ATG serum (n=4), which also significantly increased colony formation (NoC 225.7 ± 33.4) compared to 140.1 ± 9.3 post-ATG without AB (p=0.014). Furthermore, phosphorylation of phospho-SMAD2/3 was found to be significantly lower in post-ATG (2052 ± 428, mean fluorescence intensity, n=4) compared to pre-ATG (3323 ± 32; p=0.03) treated cells. This effect was partially rescued by adding EPAG to the post ATG sera (2247 ± 627, p=0.239). Finally, we analyzed whether EPAG could rescue AA BM in presence of their matching serum, pre- and post- ATG treatment (n=6). As expected for AA BM, the NoC obtained was substantially reduced. Adding post-ATG serum resulted in further impairment of their CP (NoC: 2.0 ± 1.6) compared to the pre-ATG serum (6.0 ± 6.7; p=0.04). Again, addition of EPAG to the post-ATG serum was able to rescue AA BM cells’ CP from such effect (NoC: 5.6 ± 4.6; p=0.04).
Summary/Conclusion: We provide evidence that treatment with ATG has an initial negative impact on the CP of HSC from HD and AA pts, likely mediated through acute release of IFN-γ. The addition of both IFN-y-NAB as well as of EPAG protects HSC from this effect. These results indicate a potential explanation for the superior response rates of EPAG given concurrently with ATG-based IST compared to later application and suggest that administration of EPAG should be started simultaneously with IST in order to maximize its beneficial effect in AA pts.