Modified recombinant human erythropoietin with potentially reduced immunogenicity

Recombinant human erythropoietin (rHuEPO) is a biopharmaceutical drug given to patients who have a low hemoglobin related to chronic kidney disease, cancer or anemia. However, some patients repeatedly receiving rHuEPO develop anti-rHuEPO neutralizing antibodies leading to the development of pure red cell aplasia (PRCA). The immunogenic antibody response activated by rHuEPO is believed to be triggered by T-cells recognizing EPO epitopes bound to MHC molecules displayed on the cell surface of APCs. Previous studies have reported an association between the development of anti-rHuEpo-associated PRCA and the HLA-DRB1*09 gene, which is reported to be entrenched in the Thai population. In this study, we used computational design to screen for immunogenic hotspots recognized by HLA-DRB1*09, and predicted seventeen mutants having anywhere between one through four mutations that reduce affinity for the allele, without disrupting the structural integrity and bioactivity. Five out of seventeen mutants were less immunogenic in vitro while retaining similar or slightly reduced bioactivity than rHuEPO. These engineered proteins could be the potential candidates to treat patients who are rHuEpo-dependent and express the HLA-DRB1*09 allele.


Modified recombinant human erythropoietin with potentially reduced immunogenicity
Thanutsorn Susantad 1,2 , Mayuree Fuangthong 2 , Kannan Tharakaraman 3 , Phanthakarn Tit-oon 2 , Mathuros Ruchirawat 2* & Ram Sasisekharan 3,4* Recombinant human erythropoietin (rHuEPO) is a biopharmaceutical drug given to patients who have a low hemoglobin related to chronic kidney disease, cancer or anemia. However, some patients repeatedly receiving rHuEPO develop anti-rHuEPO neutralizing antibodies leading to the development of pure red cell aplasia (PRCA). The immunogenic antibody response activated by rHuEPO is believed to be triggered by T-cells recognizing EPO epitopes bound to MHC molecules displayed on the cell surface of APCs. Previous studies have reported an association between the development of anti-rHuEpo-associated PRCA and the HLA-DRB1*09 gene, which is reported to be entrenched in the Thai population. In this study, we used computational design to screen for immunogenic hotspots recognized by HLA-DRB1*09, and predicted seventeen mutants having anywhere between one through four mutations that reduce affinity for the allele, without disrupting the structural integrity and bioactivity. Five out of seventeen mutants were less immunogenic in vitro while retaining similar or slightly reduced bioactivity than rHuEPO. These engineered proteins could be the potential candidates to treat patients who are rHuEpo-dependent and express the HLA-DRB1*09 allele.
Erythropoietin (EPO) is a protein hormone produced by the kidneys and plays an essential role in the production and maturation of red blood cells (RBCs), which carry oxygen from the lungs to the rest of the body. EPO contains 165 amino acids, which contributes to the relative molecular mass of around 30,600 daltons. Post-translational modification of this protein results in the addition of 4 carbohydrate chains: 3 N-linked and 1 O-linked glycosylation 1-3 , following which the molecular weight of erythropoietin is roughly 40% increased from its original mass. Recombinant human erythropoietin (rHuEPO) is administered to patients who have lower hemoglobin levels because of their inability to produce enough endogenous erythropoietin. These include patients having chronic kidney disease (CKD), patients dependent on dialysis, HIV infection and malignancy. rHuEPO has also been used to accelerate erythropoiesis in surgery, post-chemotherapy and post-transplantation 4 . Indeed, treatment of anemia with rHuEPO has been shown to improve the quality of life (QoL) of these patients 5,6 .
Several reports have shown that the number of reported cases with PRCA due to the development of neutralizing antibodies against endogenous EPO and recombinant erythropoiesis-stimulating agents (ESAs) has increased worldwide [7][8][9][10][11][12][13] . Most of these reported cases were in patients receiving rHuEPO for the treatment of CKD-related anemia. The anti-rHuEPO neutralizing antibodies cross-react with endogenous EPO. The development of autoantibodies against endogenous EPO in patients who have never been treated with ESAs is very rare 14 . While PRCA is a type of anemia associated with several causes including virus infection, immunological mediation, pregnancy, pre-stage malignancy, toxic exposure, and drug effects, the distinct features of PRCA associated with rHuEPO includes severe rHuEPO resistance, blood transfusion dependence, high serum ferritin, bone marrow showing the absence of red cell precursor and presence of anti-rHuEPO antibody 15,16 . PRCA symptoms include very low reticulocyte and erythroid progenitor cells while other blood cell parameters are normal 15 . The incidence of EPO-associated PRCA is a significant burden on the affected population worldwide, especially in the most affected countries such as Thailand 17 .
The immunogenic antibody response activated by rHuEPO is believed to be T-cell dependent 16,18 . An antigenpresenting cell (APC) such as dendritic cell (DC) uptakes, processes and presents antigen as a peptide epitope

Results
Designing rHuEPO with lower immunogenicity in patients with HLA-DRB1*09 gene. We carried out a computational assessment of MHC II (HLA-DRB1*09-DQB1*03:09) allele binding hotspots on EPO amino acid sequence with the idea of introducing mutations that would disrupt the binding with the HLA class II allele while not affecting the structure and binding of EPO receptor. We employed NetMHCII 2.2-a tool for HLA class II allele peptide prediction-to predict sites on EPO that would favor binding to HLA-DRB1*09-DQB1*03:09 allele 22 . The five core binding sites were predicted based on HLA-DRB1*09 (*09:01, *09:02, *09:03, *09:04, *09:05, *09:06, *09:07, *09:08, *09:09) to be VLRGQALLV (position 74-82), LRSLTTLLR (position 102-110), LLRALGAQK (position 108-116), FRVYSNFLR (position 142-150), and YSNFLRGKL (position 145-153), and this mapped to 3 distinct non-overlapping regions (residues 74-82 (VLRGQALLV), 102-116 (LRSLTTLLRALGAQK) and 142-153 (FRVYSNFLRGKL)) ( Fig. 1a). However, no significant hits were found for HLA-DQB1*03:09. Interestingly, the predicted alleles overlapping with the sites recognized by the two cellsurface erythropoietin receptors, inferred from the co-crystal structure (PDB: 1EER). Known sites of neutralizing anti-EPO antibodies from the literature were also mapped. Our intention was to modify residues that are in the predicted allele binding regions but not involved in the EPO-R binding interface (Fig. 1b). We chose to focus more on regions 102-116 and 142-153 since they overlap or in proximity to known neutralizing antibody sites. This involved a total of 25 sites: 74,75,76,77,79,80,81,82,102,105,106,109,111,112,113,114,115,116,142,145,146,148,149,152 and 153 (Fig. 1a). The design process had to factor the impact any amino acid mutation may have on the structure as allele sites considered for modification ( Fig. 1b) were located within the highly networked helices (B-C-D) of the alpha-helical bundle. Because many of the sites were buried within the core of the four helical bundle structures, we engineered networks of interacting mutations, in many cases, to preserve structural integrity while disrupting allele binding (Fig. 1). Optimization of interatomic contacts was made using a variant of the EPCN method 23 . This exercise led to a total of 17 HLA-affinity-disrupting designs on EPO (Table 1).
Characterization of the rHuEPO proteins. Seventeen HLA-affinity-disrupting mutants of EPO were expressed in FreeStyle 293-F cell line and the proteins were purified and quantitated as described in the Materials and Methods. SDS-PAGE was used together with Coomassie blue staining to reveal the protein content in the purified EPO protein from FPLC (Fig. 2a). The size of deglycosylated and glycosylated EPO protein is estimated to be 30.6 kDa and 36-40 kDa, respectively 1 . The biological reference preparation (BRP) of EPO batch 4 (European Directorate for the Quality of Medicines & Health Care European Pharmacopoeia, France) was used as reference 24 . Both BRP, purified EPO-WT and EPO mutants showed expected molecular weight (Fig. 2a). Western blot analysis using anti EPO antibody showed the positive band for all samples including BRP, EPO-WT and EPO mutant proteins (Fig. 2b), confirming the identity of the proteins.
The carbohydrate portions of EPO contain sialic acid molecules. The variability in sugar structure and the number of sialic acid molecules also affect on EPO protein that exists as a mixture of isoforms. The sialic acid residues are necessary for biological activity including receptor binding affinity and serum half-life. Removal of sialic acid from EPO showed an increased in vitro bioactivity but reduced in vivo bioactivity 25 . The isoform distribution of the products was represented by the number of bands separated on the IEF gels. Purified EPO samples were separated on an IEF gel according to their pI. The glycoform distribution among BRP and 6 EPO samples (1 wild type and 5 mutants) were observed in the basic area of the gel (Fig. 3). According to an established method (TD2014EPO) for detection and reporting of rHuEPO using an electrophoretic technique provided by WADA, BRP showed 6 isoforms as expected. However, the number of isoforms among EPO samples varied  www.nature.com/scientificreports/ distribution was varied. This might be from the differences in the microheterogeneity of glycosylation between EPO samples. However, the higher isoforms of the proteins might exhibit an increase in bioactivity due to a decrease in serum clearance, which could be administered less frequently than the original drug.
EPO mutations at the HLA-DRB1*09 core binding sites that retain its bioactivity. The bioactivity of mutated EPO proteins (Table 1) was measured in a cell proliferation assay using the TF-1 cell line. The TF-1 cell line, which expresses endogenous EPO receptors, has been derived from a patient with erythroleukemia. EPO also plays a role in the short-term growth of these cells and induces their differentiation into mature RBCs. In vitro bioactivity screening of EPO-WT and 17 mutants using TF-1 proliferation assay indicated that only 5 mutants showed bioactivity (EPO-1.2 (L70V, V74L and L1023I), EPO-3.1 (T106A), EPO-3.2 (T106G), EPO-3.3 (T106H), and EPO-4.1 (L109A)) ( Fig. 4). All 5 mutants showed a lower bioactivity than the wild-type, especially EPO-1.2 and EPO-4.1 with the relative bioactivity (REP) to EPO-WT of 0.059 (or 5.9% activity compared to EPO-WT) and 0.032 (or 3.2% activity compared to EPO-WT), respectively. EPO-3.1 had a highest bioactivity among EPO mutants with REP of 0.431 (or 43.1% activity compared to EPO-WT). EPO-3.2 and EPO-3.3 were in the same range of bioactivity with REP of 0.243 (or 24.3% activity compared to EPO-WT) and 0.229 (or 22.9% activity compared to EPO-WT), respectively.
Immunogenicity test of purified rHuEPO proteins. Next, the EPO mutants that retain bioactivity were tested for their ability to reduce immunogenicity using an ex vivo assay. Blood samples from 50 healthy volunteers were collected and expression of HLA-DRB1*09 was determined since the mutations were designed based on HLA-DRB1*09 alleles. Eleven out of fifty volunteers (22%) had HLA-DRB1*09, of which there were www.nature.com/scientificreports/ four women (36%) and seven men (64%). All HLA-DRB1*09 negative and positive volunteers were randomly selected for immunogenicity test and were confirmed to be negative for common infectious diseases including hepatitis B, hepatitis C, syphilis, and HIV. To test whether the engineered EPO proteins with disrupted HLA-DRB1*09 binding affinity were less immunogenic, PBMCs from 3 HLA-DRB1*09 negative volunteers and 3 HLA-DRB1*09 positive volunteers were isolated. The high resolution of HLA-DRB1 types of these 6 volunteers are presented in Supplementary Table S1. Precursor DCs were separated from other immune cells by adherence to plastic whereas non-adherent cells were used as a source of CD4 + T cells. Maturation of precursor DCs to immature DCs by determination of HLA-DR expression was monitored by flow cytometry using FITC-conjugated anti-human HLA-DR. Percentage of immature DC maturation of both groups ranged from 50.7 to 80.4  www.nature.com/scientificreports/ on day 7 of DC culture. To generate mature DCs as an antigen presenting cells, immature DCs were pulsed with BRP or EPO protein (EPO-WT, EPO-1.2, EPO-3.1, EPO-3.2, EPO-3.3 and EPO-4.1) for 48 h. CD4 + T cells were isolated from the nonadherent cell fraction from the same volunteer who provided PBMC. Subsequently, EPO-pulsed DCs and CD4 + T cells were cocultured at a 1:20 ratio. Since anti-CD3 and anti-CD28 antibodies are known to stimulate T cells in a manner that partially mimics stimulation by APCs 26 . Therefore, the incubation with anti-CD3/anti-CD28 antibodies was used as a positive control, which activated T cell response. IFN-γ released by CD4 + T cells as a T cell response was quantified by sandwich ELISA. Level of IFN-γ from coculture between unpulsed DC and CD4 + T cells was considered as a background of T cell response. Statistical comparison of the relative T cell response of EPO protein compared to BRP between HLA-DRB1*09-negative and positive groups demonstrated that all 5 EPO mutants had a significant lower T cell response in HLA-DRB1*09positive group. The p values were 0.00382 for EPO-1.2, 0.00002 for EPO-3.1, 0.00004 for EPO-3.2, 0.00031 for EPO-3.3 and 0.00116 for EPO-4.1.There was no difference in T cell response using EPO-WT between HLA-DRB1*09-negative and positive groups (Fig. 5a). The level of IFN-γ release from each HLA-DRB1*09-positive and negative volunteers is shown in Fig. 5b,c, respectively. In both HLA-DRB1*09-positive and negative group, BRP and EPO-WT could stimulate T cell response in the same manner as anti-CD3/anti-CD28 antibodies. There were no statistically significant differences among BRP, EPO-WT and anti-CD3/anti-CD28 antibodies in both positive and negative groups. In Fig. 5b, EPO mutants including EPO-1.2, EPO-3.1, EPO-3.2, EPO-3.3 and EPO-4.1 exhibited a lower T cell response with the IFN-γ release ranging from 220 to 37,000 pg/mL in positive group. As compared to EPO-WT, all EPO mutants showed the significant differences (p ≤ 0.0001). Figure 5c showed data from negative group; and EPO-WT and all EPO-mutants showed no significant differences in T cell response as compared to anti-CD3/anti-CD28 antibodies.
The different baseline of EPO-specific T cell response seen in each volunteer might be due to the difference in HLA allele distribution in individuals supported by 2 studies. Praditpornsilpa et al. 17  These studies clearly illustrated the antigenicity of EPO among various HLA types. In conclusion, the engineered mutants exhibit lower immunogenicity while retaining key structural characteristics and bioactivity as summarize in Table 2.
Predicted binding between EPO mutants and common HLA class II alleles. In order to assess the potential impact of the engineered mutations in a broader context, NetMHCIIpan version 3.2 was used to predict the peptide binding affinity to MHC class II molecules 22 (Table 3) 27 . In particular, 7 DR alleles are common in Southeast Asia population including DRB1*07:01, DRB1*09:01, DRB1*11:01, DRB1*12:01, DRB1*15:01, DRB1*04:05, and DRB1*03:01 19 . The predicted affinity was shown in term of a percentile rank. A percentile rank for a peptide was generated by comparing its affinity against the scores of 200,000 random natural peptides of the same length of the query peptide. A weak binder was identified if the % rank was below 25%. A strong binder was identified if the % rank was below 2%. The fragments 68-82 and 104-118 of EPO-WT had varying affinities for the various alleles, with up to 115-fold variation. Except EPO-4.1, none of the designs showed greater than two-fold variation in binding compared to EPO-WT. EPO-4.1 showed two to five-fold decrease in affinity to 9/26 alleles (DRB1*01:01, DRB1*11:01, DRB1*12:01, DRB1*15:01, DRB1*04:01, DRB1*04:05, DRB4*01:01, DRB5*01:01, DPA1*02:01-DPB1*05:01) and three to six-fold increase in affinity to only 2/26 alleles (DQA1*05:01-DQB1*03:01 and DQA1*01:02-DQB1*06:02) ( Table 3). Although, the peptides 68-82 and 104-118 of EPO-WT had appreciable affinities for DRB1*09:01 suggesting that these motifs are potential binding sites of the allele. The mutants however did not show a drop in binding, as expected, contrasting the findings of our ex vivo experiment. Collectively, the results of the in silico analysis show that (1) the engineered mutations do not pose any risk of increased immunogenicity due to the common alleles and (2) of all the designed variants, EPO-4.1 shows the highest potential to exhibit reduced immunogenicity relative to EPO-WT.

Discussion
Since the rHuEPO become more available for treatment of anemia of patients having CKD, some CKD patients who have previously or are currently using rHuEPO have been reported to display suspected or confirmed PRCA 17 . A report in 2005 indicated that 4 Thai patients who received rHuEPO therapy had a confirmed anti-rHuEPO associated PRCA and all of them also displayed HLA-DRB1*09 17 . DRB1*09 allele has a high frequency among Southeast Asian countries. The immunogenic adverse effect of rHuEPO is proven to be T-cell dependent. The disruption between EPO epitope and HLA-DRB1*09 molecule is predicted to lower immunogenicity in patients with HLA-DRB1*09. We employed computational methods to modify EPO via rational protein engineering to have reduced binding to HLA-DRB1*09.
Both EPO-WT and EPO mutants were screened for their biological activity using in vitro TF-1 proliferation assay. This cell type exhibits EPO receptor on its surface and shows commitment to erythroid lineage and has an intact Jak2/Stat5 pathway, which mediates cell proliferation in response to various growth factors including EPO 28,29 . Five out of seventeen EPO mutants (EPO-1.2, EPO-3.1, EPO-3.2, EPO-3.3 and EPO-4.1) exhibited in vitro bioactivity as compared to wild type. However, the variation in bioactivity on TF-1 cells may due to the www.nature.com/scientificreports/ differences in protein sequences, overall protein conformation and glycosylation pattern. It is well known that rHuEPO has a complex glycosylation pattern and exists in a group of different isoforms. The isoform of rHuEPO play an important role in their activity, potency and also stability 25 . It has been shown that in vivo activity has greatly decreased in non-glycosylated EPO. Fully glycosylated EPO is known to contain as many as 14 isoforms.   www.nature.com/scientificreports/ at position 109 (EPO-4.1) are considered to preserve the hydrophobic network of wild type EPO. On the other hand, the single mutants involved in EPO-3.1, EPO-3.2 and EPO-3.3 alter the native amino acid character. Polar hydroxyl-containing amino acid threonine at position 106 was changed to smaller and non-polar amino acids, which are alanine (EPO-3.1) and glycine (EPO-3.2). EPO-3.3, on the other hand, was modified to have an imidazole side chain at 106 to avoid recognition of HLA class II allele. All EPO candidates containing these mutated sequences might be considered as the altered HLA epitope that might not be recognized by the HLA molecule leading to the lower immunogenic response in patient who uses EPO and has this HLA.
To test this hypothesis, the immunogenicity of EPO protein was evaluated using PBMC from HLA-DRB1*09negative and positive volunteers. The screening of HLA-DRB1*09 in volunteers living in Bangkok, Thailand found that 22% volunteers have HLA-DRB1*09 confirming the high frequency of this particular HLA in Thailand. Because the types of MHC molecules expressed in laboratory animal are different from human, therefore, we used an ex vivo human primary model rather than animal model. Due to the small study group, apart from t-test, we also confirmed the statistically significant differences of the T cell response among EPO mutants between HLA-DRB1*09-negative and positive groups using the non-parametric Mann-Whitney U test. The statistical differences at p ≤ 0.01 were obtained for all EPO mutants compared to the EPO-WT (data not shown). The statistical differences at p ≤ 0.01 were obtained for all EPO mutants compared to the EPO-WT (data not shown). The magnitude of immunogenicity observed in all 5 EPO mutants was significantly decreased in HLA-DRB1*09-positive volunteers when compared to HLA-DRB1*09-negative volunteers. These altered proteins with reduced immunogenicity could associate with the reduced HLA-DRB1*09 binding affinity. A study by Tangri et al. identified EPO epitopes and modified those epitopes in EPO protein to reduce HLA-DR binding. The modified EPO protein showed bioactivity and lower immunogenicity 21 . The mutations reported in this paper are distinct from Tangri et. al.
The potential impact of the engineered mutations in a broader context was assessed and indicated that the engineered mutations do not pose any risk of increased immunogenicity due to the common alleles in Southeast Asia and global populations. However, the in silico prediction did not show the drop in binding affinities of the engineered mutations to DRB1*09:01 allele as compared to the ex vivo data in this study. This apparent contradiction could be due to limitations in capturing the effects of site-specific modifications on allele binding. We noted that the design of the EPO variants did not rely on comparing the score pre-and post-modification. Rather the design process was governed by several sequence and structural factors such as: (1) the location of the site to be modified within the core peptide, (2) proximity to known neutralizing antibody sites (3) whether the modification will have any impact on binding EPO receptor, and (4) whether the modification will destabilize the structure by affecting interatomic networks.
Due to a high incidence of rHuEPO-associated PRCA cases and high frequency of HLA-DRB1*09 in Thailand, here, we successfully used in silico tool to design EPO mutants to disrupt the EPO-T cell interaction. The mutation was made to reduce the binding between EPO epitope and HLA-DRB1*09 paratope. Five out of seventeen mutants exhibited biopotency confirming their unaffected biological function. A significant decrease in T cell response in HLA-DRB1*09-positive volunteers demonstrated that these engineered proteins could be the potential alternative candidates for patients who have HLA-DRB1*09 gene with no potential impact on the increased immunogenicity to the other common alleles found in Southeast Asia and global populations.
MHC epitope prediction analysis. In silico tool was used to predict amino acid residues responsible for binding between EPO epitopes and HLA class II allele HLA-DRB1*09-DQB1*03:09. The residues responsible for maintaining the structural integrity of the EPO protein and EPO receptor binding were unchanged in order to maintain its bioactivity. The non-receptor-binding site (non-RBS) residues were modified to reduce the HLA and neutralizing antibody binding. The interatomic contacts were optimized using a variant of the EPCN method 23 . Seventeen EPO mutants were designed (Table 1).

Affinity prediction to common HLA class II alleles. The binding prediction of EPO-WT and mutants
(EPO-1.2, EPO-3.1, EPO-3.2, EPO-3.3 and EPO-4.1) to the common HLA class II alleles was performed using NetMHCIIpan version 3.2 (http://www.cbs.dtu.dk/servi ces/NetMH CIIpa n-3.2/). The two regions (15-mer peptides 68-82 and 104-118) were used for a peptide scan. The common 15 DR alleles, 6 DQ alleles and 5 DP alleles were included in the analysis. NetMHCIIpan version 3.2 was run with the following settings: peptide length to scan: 15, threshold for weak binder (% rank) < 25%; threshold for strong binder (% rank) < 2%, where the percentile rank for a peptide is generated by comparing its affinity against the scores of 200,000 random natural peptides of the same length of the query peptide.
Plasmid generation and preparation. A pcDNA 3.3 expression vector containing a sequence encoding EPO mutant having only 1 mutation/substitution site was generated using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies). A sequence encoding for EPO mutant having more than 1 mutation/ substitution sites was generated using gene synthesis, cloned into pcDNA 3.3 expression vector, and transformed into E. coli (DNA 2.0). Purified plasmids were submitted for DNA sequencing (Genewiz) to confirm the mutations.

Volunteers and preparation of peripheral blood mononuclear cells (PBMCs). This study was
approved by the human research ethics committee, Chulabhorn Research Institute, Thailand. All methods involving human participants were performed in accordance with the guidelines and regulations of the institutional ethical committee. All volunteers gave a written informed consent. Blood was collected from healthy volunteers in EDTA-treated vacuum tubes (Becton Dickinson). HLA-DRB1 type was determined using sequencespecific oligonucleotide primed PCR (PCR-SSO) with the use of the Luminex technology at Faculty of Medicine, Chulalongkorn University, Thailand. Common infectious diseases including hepatitis B, hepatitis C, syphilis and HIV were also screened. PBMCs were isolated using IsoPrep solution (Robbins Scientific Corporation).
Dendritic cell (DC) culture and flow cytometric analysis. A total of 10 × 10 6 cells of PBMCs in 3 mL complete RPMI medium were plated in each well of a 6-well plate. After incubation at 37 °C under 5% CO 2 for 2 h, the nonadherent cells were collected as a source of CD4 + T cells. The complete RPMI medium containing 50 ng/mL GM-CSF and 50 ng/mL IL-4 (ImmunoTools) was added on the adherent cells representing the precursor DCs. Cells were incubated and 75% spent medium was exchanged to fresh medium with cytokines every second day. The differentiation of precursor DCs to immature DCs was monitored by a BD FACSCanto Flow cytometer (BD Biosciences) using FITC-conjugated anti-human HLA-DR (ImmunoTools).
Generation of mature DCs. Immature DCs were harvested and washed with complete RPMI medium. A total of 10 5 cells were plated in each well of a 24-well plate and incubated (pulsed) with 10 µg/mL BRP or EPO protein for 48 h. Immuture DCs incubated without protein were used as control DCs (unpulsed DCs).
Separation and activation of CD4 + T cells. CD4 + T cells were prepared from the nonadherent cell fraction of PBMCs from the same volunteer who provided monocytes for the DC culture. CD4 + T cells were isolated by negative selection using MACS Human CD4 + T cell Isolation Kit (Miltenyi Biotec). Viability was greater than 90% (data not shown). To activate effector immune cells, EPO-pulsed (mature) DCs and CD4 + T cells derived from the same volunteer were cocultured at a 1:20 ratio. Positive control included the coculture of unpulsed DC and CD4 + T cells supplemented with 5 µg/mL anti-human CD3 and 2 µg/mL anti-human CD28 (ImmunoTools). Background control included the coculture of unpulsed DC and CD4 + T cells alone. All cocultures were incubated in the presence of 30 ng/mL IL-2 and 30 ng/mL IL-12 (ImmunoTools) for 4 days and then harvested. The level of IFN-γ representing T cell response was quantified by Human IFN gamma ELISA Kit (ImmunoTools). gel. An electrophoresed gel was stained with InstantBlue protein stain (Expedeon). The purity of protein bands was analyzed by measurement of band intensity in each lane using ImageJ program 35 . The purity of the purified proteins was 94-99%.
Western blotting analysis. The proteins in SDS-PAGE gel were transferred onto nitrocellulose membrane (Bio-rad). The membrane was incubated with a rabbit polyclonal IgG to human EPO, AB-286-NA (R&D Systems) followed by a donkey derived ECL anti-rabbit IgG, peroxidase-linked species-specific whole antibody, NA934V (GE Healthcare). The signal was developed using an ECL Prime Western Blotting Detection Reagent (GE Healthcare). The chemiluminescent signal was detected using an ImageQuant LAS 4000 machine and analyzed using ImageQuant TL (GE Healthcare).
Isoform distribution analysis by IEF. BRP and purified EPO samples were buffer exchanged into MilliQ water and separated by an IEF gel with a pH gradient of 2-6 using a flat-bed electrophoresis cell-MultiphorII (GE Healthcare). The electrophoresed samples were then transferred to Armersham Hybond-P PVDF transfer membrane (GE Healthcare) using a semi-dry Novablot transferring system (GE Healthcare). After blotting, the membrane was processed as previously described in Western blot analysis.