Erythropoietin (EPO) is the main hormone that promotes proliferation and differentiation of erythroid progenitor cells via binding to its surface receptor (EPO-R). Recent studies suggest that this hormone may affect also other cell types, besides the red blood cell lineage. We have previously demonstrated that the immune system is a target of EPO; however, the direct target cells of EPO, as well as the molecular mechanisms underlying its role as an immunomodulator, are unknown. Here we present evidence for functional effects of EPO on dendritic cells (DCs), which are known to initiate the immune response. In-vivo experiments in EPO-injected mice and in transgenic mice over-expressing human EPO showed an increased splenic DC population with a higher cell surface expression of CD80 and CD86. Further analysis based on mouse models, showed that DCs derived in-vitro from bone marrow (BM-DCs) express EPO-R mRNA. In-vitro stimulation of these DCs with recombinant human EPO enhanced viability, upregulated CD80, CD86 and MHC class II and augmented the secretion of IL-12. Biochemical analysis of EPO mediated signaling in the BM-DCs showed activation of the AKT, MAPK and NF-kappaB pathways. EPO stimulation of the BM-DCs led to Tyr-phosphorylation of STAT3. The inability to detect EPO mediated activation of STAT5 in the BM-DCs, suggests that in DCs, STAT3 may play a more important role than STAT5 in EPO-R signaling. Taken together, our data support the premise that DCs are direct targets of EPO, thereby providing an insight to the immunomodulatory functions of EPO.

Erythropoietin (Epo) is the main erythropoietic hormone. Recombinant human Epo (rHuEpo) is thus used in clinical practice for the treatment of anemia. Accumulating data reveals that Epo exerts pleiotropic activities. We have previously shown an antineoplastic activity of Epo in murine multiple myeloma (MM) models, and in MM patients. Our findings that this anti-neoplastic effect operates via CD8 + T lymphocytes led us to hypothesize that Epo possesses a wider range of immunomodulatory functions. Here we demonstrate the effect of Epo on B lymphocyte responses, focusing on three experimental models: (i) tumor-bearing mice, (5T2 MM mouse); (ii) antigen-injected healthy mice; and (iii) antigen-injected transgenic mice (tg6), overexpressing human Epo. In the MM model, despite bone marrow dysfunction, Epo-treated mice retained higher levels of endogenous polyclonal immunoglobulins, compared to their untreated controls. In both Epo-treated wild type and tg6 mice, Epo effect was manifested in the higher levels of splenocyte proliferative response induced in vitro by lipopolysaccharide. Furthermore, these mice had increased in vivo production of anti-dinitrophenyl (DNP) antibodies following immunization with DNP-keyhole limpet hemocyanin. Epo-treated mice showed an enhanced immune response also to the clinically relevant hepatitis B surface antigen. These findings suggest a potential novel use of rHuEpo as an immunomodulator.See accompanying commentary: http://dx.doi. org/10.1002/eji.200737401 Introduction Erythropoietin (Epo), produced mainly in the adult kidney, is the major growth regulator of the erythroid cell lineage. Cloning of the Epo gene has led to the introduction of recombinant human Epo (rHuEpo) into clinical practice as a treatment for various anemias, including anemia related to chronic kidney disease and certain forms of cancer [1, 2]. Detection of the target receptor for Epo (EpoR) in cells other than erythroid progenitors, such as polymorphonuclear leukocytes, megakaryocytes, endothelial, myocardial and neural cells [3][4][5][6][7], suggests that Epo has other biological functions beyond erythropoiesis, and may have further potential therapeutic applications. These effects include improvement in congestive heart failure [8,9] and neuroprotection [10][11][12][13][14][15][16][17].* These authors contributed equally to this study. The idea that rHuEpo may have an important effect on the immune system, including both cellular and humoral type responses, derives from several lines of data, as recently reviewed [18]. For instance, clinical observations revealed that treatment with rHuEpo was associated with enhanced antibody production in hemodialysis patients, demonstrated for T cell-dependent antigens, such as tetanus toxoid and hepatitis B [19][20][21][22] as well as for the T cell-independent pneumococcal polysaccharide antigen [20]. Others have demonstrated enhancement of basal and mitogen-stimulated immunoglobulin production by cultured peripheral mononuclear cells (MNC) of dialysis patients treated ...

SummaryErythropoietin (Epo) is the main growth regulator of red blood cells, and recombinant human erythropoietin (rHuEpo) is thus used in clinical practice for the treatment of anaemia, primarily in kidney disease and cancer. rHuEpo treatment was found to be associated with prolonged survival of multiple myeloma (MM) patients. This clinical observation was then supported by studies on murine myeloma models. It thus appeared that rHuEpo had an anti-myeloma effect, causally related to an immunomodulatory function of rHuEpo. The present study investigated whether rHuEpo-treated MM patients acquire improved immunological functions. Treatment with rHuEpo, prescribed for anaemia that occurs in advanced disease, was associated with effects on a variety of immunological parameters and functions. This was expressed in an actual normalisation of the CD4:CD8 cell ratio, enhanced T cell phytohaemagglutinin-mediated activation and proliferation potential, T cell expression of the costimulatory CD28 and inhibitory CTLA-4 molecules, as well as reduced interleukin-6 serum values to normal levels. Furthermore, it was demonstrated that immunological abnormalities manifest in patients even in the early stages of MM. Our findings thus suggest that rHuEpo treatment might be effective in the early stages of MM, before anaemia develops. It is expected that this would boost the immune system, consequently achieving an anti-myeloma function; affecting disease progression and improving the prognosis.

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Erythropoietin (Epo), the main growth regulator of erythropoiesis, is being used to treat anemias associated with renal disease and malignancies. We have previously observed prolonged survival of patients with advanced multiple myeloma (MM) who were treated with recombinant human Epo (rHuEpo) (Mittelman et al, Eur J Haematol2004:72:155). Further studies on murine MM models confirmed this observation and suggested that Epo has an anti-MM immunomodulatory effect (Mittelman et al. Proc Natl Acad Sci USA2001:98:5181; Katz et al. Acta Haematol2005:114:177). We have reported (ASH 2004) preliminary results showing that the rHuEpo-treated MM patients acquired improved immunological functions. We have since then extended the numbers of patients and the range of immunological functions tested. Here, we show that rHuEpo, prescribed for anemia in patients with advanced MM (Durie and Salmon Stage 2–3), is associated with effects on a variety of immunological parameters and functions, as presented in the table. Compared with non rHuEpo-treated MM patients, who demonstrated immunological abnormalities, rHuEpo-treated MM patients resumed normalization of the CD4:CD8 cell ratio, enhanced T-cell (both CD8 and CD4) PHA-mediated activation, improved mononuclear proliferation potential, higher expression of the co-stimulatory molecule CD28, lower values of the inhibitory CTLA-4 molecule and decreased levels of serum IL-6. The immunological parameters in the rHuEpo-treated MM patients were close to the normal healthy controls. Results are presented in the table below (mean +/− SE). We also found similar immunological abnormalities in patients at an early stage (Durie and Salmon stage 1, smoldering) MM (n=8). Our findings a) confirm and extend reports by others regarding immunological abnormalities in patients with advanced MM; b) show that patients with advanced MM treated with rHuEpo for their anemia benefit from improved immunological functions; c) show that patients with early-stage MM already manifest similar immunological abnormalities. Our data thus indicate that patients with early-stage MM might benefit from rHuEpo with improved immune functions, even prior to the development of anemia in later stages of the disease. Taken together, our study suggests rHuEpo as a potential immunomodulatory agent in the treatment of MM. Parameter Normal Healthy Controls MM Patients rHuEpo-treated Advanced MM Patients Early MM Advanced MM a. p<0.05 advanced MM compared to healthy controls. b. p<0.05 early MM compared to health controls. c. p<0.05 rHuEpo treated advanced MM compared to non-treated advanced MM patients Number of Patients N=14 N=8 N=21 N=13 CD4:CD8 2.4±0.3 1.2±0.2b 1.2±0.1a 1.9±0.3c PHA act- CD8+CD69+ (%) 60±4.4 45±4.4b 46.3±3a 63.3±3.8c PHA act-CD4+CD69+ (%) 57.1±3.8 42±4.6b 43.3±3a 59.9±3.6c Proliferation (%) 232.5±10.3 161±7.1b 146.5±15.1a 218.1±22.5c CD8+CD28+ (%) 73.2±5.2 53.7±8.2 38.8±2a 55.6±2.9c CD8+CTLA-4+ (%) 2.4±0.6 12.5±3.6 10.1±1.5a 4.6±0.9c sIL-6 (pg/ml) 2.6±0.4 5.4±1 10±2.1a 5.7±0.9c

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1637 Poster Board I-663 Introduction Recombinant human erythropoietin (EPO) has been effective in treating various types of anemia. EPO receptors on non-erythroid cells suggest that EPO may also have non-erythroid effects. Indeed, we have previously shown that EPO has anti-neoplastic immunomodulating effects in both patients and mice (Mittelman PNAS 2001; Mittelman Eur J Haematol 2004). EPO effects were demonstrated in both the cellular and humoral immune systems (Katz Acta Haematol 2005; Katz Eur J Immunol 2007; Prutchi-Sagiv Br J Haematol 2006; Prutchi-Sagiv Exp Hematol 2008; Lifshitz Mol Immunol 2009). Hassan et al. (Ren Fail 2003) have reported that patients with renal failure treated with EPO had a higher antibody (Ab) titer in response to the Hepatitis B vaccine. We therefore hypothesized that EPO plays a general role in augmenting the immune response. To test this hypothesis we examined the effect of EPO on the antibody response to the seasonal influenza (flu) vaccine. Methods Three groups, all receiving the flu vaccine, were tested: A) Healthy controls (HC), B) Hematologic patients not receiving EPO (H-NoEPO), C) Hematologic patients on EPO for anemia (H-EPO). Mean ages of the participants [95% CI] were 59.5 [56.5-62.4], 59.6 [50.9-68.2], and 73.9 [68.8-79.0] respectively. Flu vaccine and EPO were ordered by the attending physicians only, irrespective of the study. Blood samples were drawn prior to and 3 weeks, 7 weeks and 4 months after the flu vaccination. Anti-flu Ab titer was measured using the complement fixation test. The results are presented as the number (percentage) of tested individuals who responded with at least two- or four-fold increase in anti-flu titers, compared to their baseline. P value represents comparison of H-EPO to H-NoEPO. Results Of the HC individuals, 90% doubled (at least) and 58% quadrupled their anti-flu Ab titers. Of the H-NoEPO patients only 56% doubled and 19% quadrupled their titers. However, 72% of the H-EPO patients doubled and 56% quadrupled their titers, a response approaching that of healthy controls, despite their being significantly older. Conclusions 1) Hematologic patients respond poorly to the flu vaccine, compared to healthy subjects. 2) EPO treatment is associated with an improved immune response to the flu vaccine in hematologic patients. This suggests that EPO may serve as an immune augmenting agent for the flu vaccine and possibly other vaccines as well. Disclosures Off Label Use: Non erythroid effects: immune, anti-cancer (all under investigation). Prutchi-Sagiv:BioGAL- Start up (inactive): Equity Ownership. Mittelman:BioGAL- Start up (inactive): Equity Ownership, Patents & Royalties.