Immune Dysfunction in Patients with MDS Is Partially Corrected in EPO-Treated Patients: Differences According to WHO Classification
Authors: Oster Howard S , Deshet-Unger Naamit , Neumann Drorit , Mittelman Moshe , Maaravi Nir , Prutchi-Sagiv Sara , Nataliya Golishevski 
Published: October 2019

Abstract
Background: The immune system has been shown to be involved in the pathogenesis of myelodysplastic syndromes (MDS), and is also affected by the disease. Recombinant erythropoietin (rHuEPO), or in general, erythroid stimulating agents (ESAs) have become a standard treatment for anemic patients with MDS. They were found to improve anemia, quality of life, and possibly survival. We have previously demonstrated that EPO has effects on cellular and humoral immunity and specifically, on immune function in patients with multiple myeloma (MM). Here we report our findings demonstrating the effect of ESAs on T cell (CD4+, CD8+ and CD4+CD25+) number and function in patients with MDS.
Patients and Methods: We examined three groups: healthy subjects ('Control', 20 participants), MDS patients without ESA treatment ('MDS', 13), and MDS patients treated with an ESA ('MDS+EPO', 17). All diagnosed patients gave informed consent as approved by our IRB. Cell numbers were evaluated with flow cytometry. In a subset of patients, cell activation was assessed in response to phytohemagglutinin (PHA) by examining CD69 expression in both CD4+ and CD8+ cells. The co-stimulatory marker, CD28, and the inhibitory marker CTLA-4 (CD152) were evaluated as well. We also examined World Health Organization (WHO) subgroups, refractory anemia (RA) and RA with ringed sideroblasts (RARS) versus more advanced disease.
Results: CD4+ and CD8+ T cell levels are reduced and increased respectively in MDS patients compared to control, and these changes are reversed in MDS+EPO (Table 1, CD4+, p<0.01; CD8+, p=0.05). The CD4+:CD8+ ratio (Table 1) is reduced and nearly equalized in MDS (1.16), but approaches that of the control (2.24) in MDS+EPO (1.94). CD4+CD25+ T cell numbers (including regulatory T cells), were lower in MDS patients and improve in the MDS+EPO group (Table 1). In vitro activation of T cells (CD4+CD69+ and CD8+CD69+) achieves an approximately 15-fold increase in healthy subjects. MDS patients without EPO sustained only a 7.17 fold increase in CD4+ activation versus 13.64 fold for the MDS+EPO group (p<0.01); for CD8+ T cells, 10.20 fold (MDS) versus 18.56 fold (MDS+EPO, p<0.01). The expression of the co-stimulatory marker CD28 was decreased in both CD4+ and CD8+ T cells in MDS, and approached normal in MDS+EPO in CD4+ T cells (Table 1). There was no significant change in inhibitory CTLA-4 (CD152) expression among the groups (not shown). Subgroup analysis demonstrated that ESA has a similar effect on CD4+ and CD8+ cells and their ratio in both RA/RARS and more advanced disease, similar to those of the whole cohort (Table 2, green). On the other hand, some parameters were affected by ESA only in one subgroup (Table 2, blue): The ESA effect on CD4+CD25+ cells was evident only in patients with advanced disease (Table 2, blue). ESA affected CD4+ and CD8+ cell stimulation (CD69) in RA/RARS, similar to that seen in the whole cohort (Table 2, blue). Of note, in more advanced disease, CD4+ and CD8+ cells achieved stimulation in the MDS group not treated with ESA, with no difference between MDS and MDS+EPO. This finding needs to be further addressed in larger cohorts and with additional markers of activation.
Conclusions: MDS patients display T-cell abnormalities that are improved upon EPO treatment. MDS is a heterogeneous disease where the immune system both affects and is affected by the disease. As such, treatment with ESAs might ameliorate not only the anemia, but also the immune deficiencies and perhaps the disease itself. Future studies will clarify the immunomodulatory role of ESA in the various stages of MDS.

Disclosures
No relevant conflicts of interest to declare.