Anti-Leukemic Effects Induced by Dendritic Cells of Leukemic Origin from Leukemic Blood Samples Are Comparable under Hypoxic vs. Normoxic Conditions

Simple Summary Dendritic cells are important mediators in the activation of the innate and adoptive immune response. The generation of DC/DCleu was comparable under physiological hypoxic and normoxic culture conditions, with no significant differences in frequencies of generated DC/DCleu from leukemic cell lines, peripheral blood mononuclear as well as whole blood cells from patients with acute myeloid leukemia. Moreover, the composition of immune cells and anti-leukemic immune activity after T cell enriched mixed lymphocyte culture with patients’ blood pretreated vs not pretreated with defined response modifiers (Kits) was improved, particularly under hypoxic conditions. These data show that the generation of DC/DCleu as well as the induction of anti-leukemic immune activating functionality is possible under standard normoxic as well as physiological hypoxic culture conditions ex vivo. Abstract Hypoxia can modulate the immune system by affecting the function and activity of immune cells, potentially leading to altered immune responses. This study investigated the generation of leukemia-derived dendritic cells (DCleu) from leukemic blasts and their impact on immune cell activation under hypoxic (5–10% O2) compared to normoxic (21% O2) conditions using various immunomodulatory Kits. The results revealed that DC/DCleu-generation was similar under hypoxic and normoxic conditions, with no significant differences observed in frequencies of generated DC/DCleu. Furthermore, the study showed that the activation of immune cells and their anti-leukemic activity improved when T cell-enriched immunoreactive cells were co-cultured with DC/DCleu which were generated with Kit I and M compared to the control after mixed lymphocyte cultures. The anti-leukemic activity was improved under hypoxic compared to normoxic conditions after MLCWB-DC Kit M. These findings suggest that DC/DCleu-cultures of leukemic whole blood with Kits under hypoxic conditions yield comparable frequencies of DC/DCleu and can even increase the anti-leukemic activity compared to normoxic conditions. Overall, this research highlights the potential of utilizing DC/DCleu (potentially induced in vivo with Kits) as a promising approach to enhance immune response in patients with acute myeloid leukemia.


Introduction
Acute myeloid leukemia (AML) is a clonal hematological disorder with an overall five-year survival rate in elderly patients of about 28% [1].Although 60-80% of all patients achieve a complete remission (CR), 70-80% of these patients relapse in the following 2 years [2].Ongoing clinical trials analyze new immunotherapeutic strategies and immunomodulatory approaches to target leukemic cells, to stabilize remissions and to improve the outcome of patients with AML [3,4].
Hematopoietic stem cells, leukemic cells as well as immune cells reside in the bone marrow (BM) under hypoxic O 2 concentrations of about 0.1-1%, in the arterial blood of about 12% or of 4-15% in peripheral blood (PB) [20,21].That means that hematopoietic cells are exposed to changing O 2 concentrations/saturation during their differentiation [22].It was shown that hypoxic conditions might change the anti-leukemic activity of cells against leukemic blasts [23].Moreover, reactions of immune cells prepared ex vivo for adoptive transfer under normoxic conditions might not reflect the in vivo situation [22] and might be switched to cell preparations under hypoxic conditions.
The aim of this study was to analyze the impact of hypoxic conditions on the generation of DC/DC leu from leukemic and healthy PBMNCs and WB and furthermore, to analyze the effect of DC/DC leu stimulation on the anti-leukemic immune activation after MLC in comparison to normoxic conditions.

Sample Collection
After obtaining written informed consent in accordance with the local Ethics Committee (Pettenkoferstraße 8a, 80336 Munich, Ludwig Maximilians University Hospital in Munich; Vote No 339-05), patients' PB or BM samples were provided by the University Hospital of Oldenburg, Tübingen and Augsburg.Anticoagulation was performed with Lithium heparintubes (7.5 mL, Sarstedt, Nuernberg, Germany) containing standardized concentrations of Heparin.PBMNCs were separated by density gradient centrifugation (density gradient 1.077 g/mL) using the Ficoll Hypaque technique.CD3 + T cells were positively selected by using the MACS technology (Milteney Biotech, Bergisch Gladbach, Germany) according to manufacturers' instructions.The purity of the resulting T cells was on average 90% (range 81-97%).Cells were frozen at −80 • C (using DMSO) and thawed according to standardized protocols.
Quantification of NK cells and monocytes was not possible in cases with aberrant expression of CD14 or CD 56 on leukemic blasts.

Generation of DC/DC leu from AML Cell Lines
DC/DC leu were generated from AML cell lines with the DC/DC leu -generating protocols Kit I and Kit M [6,25].Therefore, 3-4 × 10 6 cells were pipetted in 12-multiwell plates in 2 ml serum-free X-Vivo15-medium (Lonza, Basel, Swiss) and were plated into 12-multiwell plates (Thermo Fisher Scientific, Darmstadt, Germany).Response modifiers and immune modulating factors were added to cultures as described below.A culture without added response modifiers served as a control.All response modifiers used for the DC/DC leu generation are approved for human treatment.Compositions of DC/DC leu -generating protocols are given in Table 2.
2.6.Generation of DC/DC leu from Isolated PBMNC or WB DC/DC leu were generated from 3-4 × 10 6 PBMNC (isolated by density gradient centrifugation) from healthy donors and AML patients' blood using Pici-PGE1 and Pici-PGE2 (Table 2) as described before [6,9,25,26].PBMNCs were diluted in 2 ml serum-free X-Vivo15medium (Lonza, Basel, Swiss) and were plated into 12-multiwell plates (Thermo Fisher Scientific, Darmstadt, Germany).Response modifiers were added as described below.Half medium exchange was carried out after 3-4 cell culture days.A culture without added response modifiers served as a control.
DC/DC leu were generated from healthy and leukemic WB (presenting the physiological cellular and soluble composition of the individual samples) with the DC/DC leugenerating protocols Kit I and Kit M, as described before [6,25].A culture without added re-sponse modifiers served as a control.All response modifiers used in Kits for the DC/DC leu generation are approved for human treatment.Compositions of DC/DC leu -generating protocols are given in Table 2.
2.6.3.Kit I DC/DC leu were generated with Kit I from WB and cell lines using 800 U/mL GM-CSF and 10 µg/mL Picibanil [25].After 2-3 days, the same amounts of response modifiers were added and after in total 7-10 days of incubation, cells were harvested and used for subsequent experiments.
2.6.4.Kit M DC/DC leu were generated with Kit M from WB and cell lines using 800 U/mL GM-CSF and 1 µg/mL PGE 1 [25].Incubations were performed in analogy to Kit I.
For analysis and quantification of DCs and DC leu in the total-or in the cell-subtype fractions after DC/DC leu -cultures, we used a refined gating strategy, as shown before [8,9,25].DC leu are characterized by the simultainous expression of DCs' together with patients' blast antigens.To quantify generated DC leu , the cells were stained with patients or leukemic cell line specific blast-staining antibodies (e.g., CD34, CD65, and CD117), according to diagnostic reports in combination with DC-staining antibodies (e.g., CD80, CD83, CD86, CD206, and CD209), which were not expressed on blasts before culture.DC leu were quantified in the total cell fraction (DC leu /PBMNC or WB), in the DC-fraction (DC leu /DC + ) or in the blast fraction to quantify the amounts of blasts converted to DC leu (DC leu /bla + ).Proliferating blasts were characterized by their co-expression of CD71 or IPO-38 without co-expression of DC-markers (Table 3).According to their expression profiles we quantified frequencies of immune-reactive cells in the total cell fraction (e.g., CD3 + /cells) or in the subpopulations (e.g., in CD3 + cells) as given in Table 3.

Mixed Lymphocyte Culture (MLC) of T Cell Enriched Immune Reactive Cells with Kit Treated vs Untreated WB from AML-Patients
A total of 1 × 10 6 (thawn) autologous CD3 + T cells from AML patients were cocultured with IL 2 and a stimulator cell suspension containing approximately 2.5 × 10 5 generated DC/DC leu which were generated with different DC/DC leu -generating protocols from leukemic WB.MLC of T cell enriched immunoreactive cells with a stimulator cell suspension without pretreatment with different DC/DC leu -generating protocols (MLC WB ) served as a control, as shown before [28].
Cells were harvested after 6-7 days; subtypes were quantified by flowcytometry and used for cytotoxicity fluorolysis assay [28].PBMNC peripheral blood mononuclear cells; WB whole blood; CD cluster of differentiation; DC dendritic cells; DC leu dendritic cells of leukemic origin; Bla leukemic blasts; MLC mixed lymphocyte culture.

Cytotoxicity Fluorolysis Assay (CTX)
The Cytotoxicity Fluorolysis Assay was conducted to assess the lytic activity of autologous T cell-enriched immunoreactive cells after stimulation with DC/DC leu containing cell fractions after treatment with Kit M, Kit I or control (without added response modifies) after MLC ('effector cells') against autologous leukemic blasts ('target cells').Therefore, effector and target cells (with a ratio of 1:1) were co-cultured under hypoxic and normoxic conditions and incubated for 3 and 24 h.Target cells were stained with respective antibodies before incubation.After harvest, 7AAD and a defined number of Fluorosphere beads (Beckman Coulter) were added.As a control, effector and target cells were cultured separately and mingled shortly before measurements.Flow cytometric analyses were performed after 3 and 24 h of effector and target cells' co-incubation using a refined gating strategy [30].The lytic activity against leukemic target blasts (blast lysis) is defined as the difference in frequencies of viable blasts in the effector target cell cultures as compared to controls.Improved blast lysis is defined as the difference in proportions of blast lysis achieved after MLC with DC/DC leu generated with Kits compared to control.

Cell Cycle Experiments
The cell cycle profile of cell line samples was determined by staining of DNA with PI-fluorescent dyes.PI intercalates into the groove of double-stranded DNA producing a highly fluorescent signal.As PI can also bind to double-stranded RNA, the cells must be treated with RNAse for DNA resolution.We associated a fixation (paraformaldehyde) with PI staining.The PI staining of DNA allowed the detection of cells in G0/G1, S phase, and G2/M as described earlier [31].

Quantitative PCR (Real Time PCR)
Total RNA was isolated from 10 6 cells of each cell line using MagNA Pure LC mRNA HS Kit (Roche, Basel, Switzerland) according to the manufacturer's instructions.cDNA was synthesized from 1 µg aliquots of total RNA in a 20 µL standard reaction mixture using SuperScript ® III First-Strand Synthesis System for RT-PCR (Invitrogen, Camarillo, CA, USA) according to manufacturer's instructions.Quantitative Real-time polymerase chain reaction (RT-PCR) was performed using the 7900HT Fast Real-Time PCR System (Applie Biosystems, Waltham, MA, USA) with 2 µL of cDNA, Fast SYBR ® Green Master Mix (Applied Biosystems, Waltham, MA, USA) [32].We checked the expression of the fusion genes related to each cell line in Normoxic vs Hypoxic condition.Furthermore, glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as a reference for the normalization of ∆CT values.

Statistical Methods
Mean ± standard derivations are given.Statistical comparisons of two groups were performed using the two-tailed t-test (in cases with data normally distributed) and the Mann-Whitney-Wilcoxon Test (in cases with data not normally distributed).Statistical analyses were performed with Microsoft Excel 2010 ® (Microsoft, Redmond, Washington, USA) and SSPS Statistic 24 software © (IBM, Armonk, NY, USA).Pearson correlation tests were used to evaluate correlations between traits represented in graphs.Differences were defined as 'not significant' in cases with p-values > 0.1, as 'tendentially significant' (significant *) with p-values between 0.1 and 0.05, as 'significant' (significant **) with p-values between 0.05 and 0.005 and as 'highly significant' (significant ***) with p-values < 0.005.Figures were created with GraphPad Prism7 © (GraphPad Software, San Diego, CA, USA).

Prolog
In a first step, we generated DC/DC leu from four different leukemic cell lines and evaluated mRNA profiles under hypoxic and normoxic conditions.In the next step, we generated DC/DC leu from leukemic and healthy PBMNCs as well as from WB, to simulate physiological conditions with different DC/DC leu -generating protocols.Furthermore, we analyzed the immune stimulating effect of these generated DC/DC leu after MLC under hypoxic and normoxic conditions, evaluated the resulting anti-leukemic activity and correlated these findings with frequencies of DC/DC leu -subtypes.

AML Cell Lines' Phenotypic and Genotypic Profiles Do Not Change under Hypoxic Culture
We compared the relative mRNA expression levels of cell line specific fusion genes as mentioned in Table 1 after five passages of AML cell lines under hypoxic and normoxic conditions.Levels of mRNA expression were analyzed by quantitative real-time RT-PCR, using glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) as the housekeeping control.Our results indicated no significant differences for the expression of these fusion genes under hypoxic vs. normoxic conditions (NB-4 ∆∆CT 3.5 vs. 2.9; Mono-Mac-6 ∆∆CT 23.1 vs. 27.6;KG ∆∆CT 19.6 vs 20.3; THP-1 ∆∆CT 101.2 vs. 114.5)(Figure 1A).We compared the relative mRNA expression levels of cell line specific fusion genes as mentioned in Table 1 after five passages of AML cell lines under hypoxic and normoxic conditions.Levels of mRNA expression were analyzed by quantitative real-time RT-PCR, using glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) as the housekeeping control.Our results indicated no significant differences for the expression of these fusion genes under hypoxic vs normoxic conditions (NB-4 ΔΔCT 3.5 vs 2.9; Mono-Mac-6 ΔΔCT 23.1 vs 27.6; KG ΔΔCT 19.6 vs 20.3; THP-1 ΔΔCT 101.2 vs. 114.5)(Figure 1A).Results show no differences between the expression of fusion genes (PML-RARA fusion gene in the NB-4 cell line, FGFR1OP2-FGFR1 fusion gene in the KG-1 cell lines, KMT2A-MLLT3 fusion gene in the Mono-Mac-6 cell line and KMT2A-MLLT3 fusion gene in the THP-1 cell line) under hypoxic (left side) or normoxic (right side) conditions.Relative expressions of fusion genes specific for each cell line were tested after five passages.The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as the housekeeping control.The y-axis shows the ∆∆CT differences between the genes of interest and the housekeeping gene.Average frequencies ± standard deviation are given.(B).DC/DC leu subsets after DC/DC leu -generation Results of DC/DC leu subsets after DC/DC leugeneration from four different cell lines lines (NB-4, Mono-Mac-6, KG-1, and THP-1) with Kit I, Kit M and control (culture without added response modifiers) are given.Only in the NB-4 cell line could significantly higher frequencies of mature DCs be generated with Kit I and Kit M under hypoxic compared to normoxic conditions.For all other subsets, comparable frequencies could be generated.Average frequencies ± standard deviations are given.(C).Cell cycle analysis Cells were fixed in ethanol, stained with propidium iodide (PI) and analysed for DNA content to determine populations in G1 and S phases of the cell cycle.Significantly lower frequencies of cells are found in the Sphase under hypoxic vs. normoxic conditions for the NB-4, KG-1 and THP-1 cell lines.Average frequencies ± standard deviation are given.

DC/DC leu Generation from Leukemic Cell Lines with Kits Is Comparable under Hypoxic and Normoxic Conditions without Induction of Blast Proliferation
DC/DC leu -generation with Kit I and Kit M was comparable in all four leukemic cell lines under hypoxic as well as normoxic conditions with significantly higher frequencies of DC/DC leu -subtypes found after DC/DC leu -generation with Kits compared to control.In general, Kit I was superior to Kit M in generating DC/DC leu (including subgroups) from leukemic cell lines (Figure 1B).In the direct comparison of hypoxic and normoxic conditions, significantly more mature DCs (DC mat /DC + ) could be generated in the NB-4 cell line with Kit I and Kit M under hypoxic compared to normoxic conditions (Figure 1B).Also, significantly more DC leu /Bla + could be generated with Kit M under hypoxic compared to normoxic conditions.For the other cell lines, no significant differences could be found in the direct comparison of the amounts of generated DC/DC leu as well as DC/DC leu subgroups.

Significantly Lower Frequencies of Cells from Leukemic Cell Lines Found in S-Phase of Cell Cycles under Hypoxic Compared to Normoxic Conditions
We found significantly lower frequencies of cells in the S-phase of the cell cycle under hypoxic compared to normoxic conditions for the cell lines NB-4, KG-1 and THP-1.For the cell line Mono-mac-6, no significant differences were found.Frequencies of cells in the remaining cell cycle phases did not differ significantly (Figure 1C).

Generation of DC/DC leu from Leukemic and Healthy PBMNCs Is Comparable under Hypoxic and Normoxic Conditions
DC/DC leu were generated from healthy and leukemic PBMNCs (n = 9) under hypoxic and normoxic conditions with the two DC/DC leu -generating protocols Pici -PGE1 and Pici -PGE2 compared to control.The following patients' blood samples were included in this analysis: Pat.15,16,17,19,20,21,22,24,25.Further characteristics of the patients and stages of the disease are given in Table 1.Significantly higher frequencies of DC/DC leu (including subgroups) were found after culture with Pici-PGE1 and Pici-PGE2 under hypoxic as well as normoxic conditions compared to control (Figure 2).Comparable results were found for healthy cell samples.

Generation of DC/DCleu with Kits from Leukemic and Healthy WB Is Compara Hypoxic and Normoxic Conditions-without Induction of Blasts' Proliferation
We defined a cut-off value of ≥ 5% DC + /WB as a 'sufficient DC-gen leukemic WB with Kits.Under hypoxic conditions, a sufficient DC-ge possible in 88% of cases with Kit I, in 85% of cases with Kit M and in 18% of (Figure 3A).Under normoxic conditions, an adequate and sufficient DC-gen leukemic WB was possible in 96% of cases cultured with Kit I, in 77% of cas and in 25% in the control group (Figure 3A).Comparisons showed n Comparable frequencies of DC/DC leu could be generated with Pici-PGE1 and Pici-PGE2 from leukemic PBMNCs under hypoxic and normoxic conditions.Compositions of Pici-PGE1 and Pici-PGE2 protocols are given in Table 2. Average frequencies ± standard deviations of DC/DC leu and their subtypes under hypoxic (left side) and normoxic (right side) conditions are given.

Significantly Lower Frequencies of Treg Cells Found after MLC WB-DC Kits under Hypoxic Conditions Compared to Control
In general, we found a significantly higher activation status of immunoreactive T cell subtypes after MLC WB-DC Kits compared to MLC WB (e.g., Tprol-eraly, Tnon-naiv) and reduced frequencies of Treg.Frequencies of Tregs significantly decreased after MLC WB-DC Kit M and MLC WB-DC Kit I vs control under hypoxic conditions (Figure 4A).Furthermore, we found significantly higher frequencies of NK cells after MLC WB-DC Kit M compared to the control, pointing to a stimulating effect of generated DC/DCleu on cells of the innate immune system under hypoxic and normoxic conditions (Figure 4B).
In the direct comparison of hypoxic and normoxic conditions no significant difference were observed.A detailed analysis revealed that we generated significantly higher frequencies of DC (including subtypes) from leukemic WB with Kits compared to control-under hypoxic as well as under normoxic conditions (Figure 3B).Comparable results were found for healthy samples.

Significantly Lower Frequencies of T reg Cells Found after MLC WB-DC Kits under Hypoxic Conditions Compared to Control
In general, we found a significantly higher activation status of immunoreactive T cell subtypes after MLC WB-DC Kits compared to MLC WB (e.g., T prol-eraly , T non-naiv ) and reduced frequencies of T reg .Frequencies of T regs significantly decreased after MLC WB-DC Kit M and MLC WB-DC Kit I vs control under hypoxic conditions (Figure 4A).

R PEER REVIEW
13 of 21 T cells enriched immunoreactive cells were stimulated with Kit I or Kit M pretreated (DC/DCleu containing) WB compared to the control without pretreatment.Average frequencies of stimulated cells after MLC WB-DC Kit I , MLC WB-DC Kit M and MLC WB (control) ± standard deviation are given in Figure 4A.Frequencies of iNKT and NK cells under hypoxic and normoxic conditions are given in Figure 4B.Explanations of all analyzed immunoreactive cells subtypes are given in Table 3.

Kit Pre-Treated Leukemic WB, Leads to Significantly Improved Anti-Leukemic Activation after T Cell Enriched MLC, Especially in Hypoxic Conditions
We analyzed the blast lytic effect of immunoreactive cells after T cell enriched MLC WB- DC Kits and MLC WB under hypoxic as well as normoxic conditions.Blast lysis was evaluated after 3 h and 24 h of incubation of blast targets with effector cells after MLC.
After 24 h of incubation of target cells with effector cells, blast lysis was achieved in 72% of cases after MLC WB-DC Kit I , in 84% after MLC WB-DC Kit M vs 38% of cases in the control group (MLC WB ) under hypoxic conditions.Compared to the control group, significantly more cases achieved blast lysis after MLC WB-DC Kit M (Figure 5A).We could see a clear advantage in the improvement of blast lysis after MLC WB-DC Kit M compared to the control (Figure 5B) and in the frequency of improved lysis after 24 h although differences were not significant under hypoxic and normoxic conditions (Figure 5C).Furthermore, we found significantly higher frequencies of NK cells after MLC WB-DC Kit M compared to the control, pointing to a stimulating effect of generated DC/DC leu on cells of the innate immune system under hypoxic and normoxic conditions (Figure 4B).
In the direct comparison of hypoxic and normoxic conditions no significant difference were observed.
T cells enriched immunoreactive cells were stimulated with Kit I or Kit M pretreated (DC/DC leu containing) WB compared to the control without pretreatment.Average frequencies of stimulated cells after MLC WB-DC Kit I , MLC WB-DC Kit M and MLC WB (control) ± standard deviation are given in Figure 4A.Frequencies of iNKT and NK cells under hypoxic and normoxic conditions are given in Figure 4B.Explanations of all analyzed immunoreactive cells subtypes are given in Table 3.

Kit Pre-Treated Leukemic WB, Leads to Significantly Improved Anti-Leukemic Activation after T Cell Enriched MLC, Especially in Hypoxic Conditions
We analyzed the blast lytic effect of immunoreactive cells after T cell enriched MLC WB-DC Kits and MLC WB under hypoxic as well as normoxic conditions.Blast lysis was evaluated after 3 h and 24 h of incubation of blast targets with effector cells after MLC.
After 24 h of incubation of target cells with effector cells, blast lysis was achieved in 72% of cases after MLC WB-DC Kit I , in 84% after MLC WB-DC Kit M vs 38% of cases in the control group (MLC WB ) under hypoxic conditions.Compared to the control group, significantly more cases achieved blast lysis after MLC WB-DC Kit M (Figure 5A).We could see a clear advantage in the improvement of blast lysis after MLC WB-DC Kit M compared to the control (Figure 5B) and in the frequency of improved lysis after 24 h although differences were not significant under hypoxic and normoxic conditions (Figure 5C).Achieved blast lysis after MLC WB-DC Kit I , MLC WB-DC Kit M and MLC WB (control) after 24 h of co-cultures with leukemic blats (target cells).Percentage of cases with lysis after 24 h (Figure 5A), percentage of cases with improved lysis after 24 h (Figure 5B) and percentage of blasts increased/lysed after 24 h (Figure 5C) under hypoxic and normoxic conditions are presented.

Significant Correlation of Anti-Leukemic Reactivity and DC/DCleu Subtypes under Hypoxic as Well as Normoxic Conditions
Correlating improved anti-leukemic reactivity of immune reactive cells after MLC WB- DC Kit I and MLC WB-DC Kit M vs MLC WB (control) with frequencies of generated DC and DCleu in cultures with Kits, we found significant positive correlations with generated DC + /WB under hypoxia and normoxia (r = 0.7; p < 0.01; r = 0.5; p < 0.02) and with DCleu/WB under Hypoxia (r < 0.6; p < 0.05), but not under normoxia with Kit M. (Figure 6).These correlations were not found for results obtained with Kit I. Achieved blast lysis after MLC WB-DC Kit I , MLC WB-DC Kit M and MLC WB (control) after 24 h of co-cultures with leukemic blats (target cells).Percentage of cases with lysis after 24 h (Figure 5A), percentage of cases with improved lysis after 24 h (Figure 5B) and percentage of blasts increased/lysed after 24 h (Figure 5C) under hypoxic and normoxic conditions are presented.

Significant Correlation of Anti-Leukemic Reactivity and DC/DC leu Subtypes under Hypoxic as Well as Normoxic Conditions
Correlating improved anti-leukemic reactivity of immune reactive cells after MLC WB-DC Kit I and MLC WB-DC Kit M vs MLC WB (control) with frequencies of generated DC and DC leu in cultures with Kits, we found significant positive correlations with generated DC + /WB under hypoxia and normoxia (r = 0.7; p < 0.01; r = 0.5; p < 0.02) and with DC leu /WB under Hypoxia (r < 0.6; p < 0.05), but not under normoxia with Kit M. (Figure 6).These correlations were not found for results obtained with Kit I.

DC/DCleu-Based Immunotherapy for AML
AML is a clonal hematopoietic disorder with a high risk of relapse due to blasts' immune escaping mechanisms, such as impaired or mistaken antigen expressions, resistance to apoptosis or other inhibitory mechanisms [33].To address this issue, various immunotherapeutic strategies (including DC/DCleu-based strategies) are being explored to reactivate the antitumor immune response [34,35].On the one hand DCs can be generated ex vivo from monocytes and loaded with different leukemia associated antigens and on the other hand DC/DCleu can be generated directly from leukemic blasts presenting patients' individual antigen repertoire [9,10,36].Re-administration of these ex vivo generated and prepared DC/DCleu have shown to increase the frequencies of leukemia-specific (T) cells in vivo and the treatment achieved stable complete remissions in elderly AML patients [37][38][39].A new and interesting approach could be to induce the production of DC/DCleu from leukemic blasts in vivo after the treatment of patients with DC/DCleu-inducing response modifiers [6].AML is a clonal hematopoietic disorder with a high risk of relapse due to blasts' immune escaping mechanisms, such as impaired or mistaken antigen expressions, resistance to apoptosis or other inhibitory mechanisms [33].To address this issue, various immunotherapeutic strategies (including DC/DC leu -based strategies) are being explored to reactivate the antitumor immune response [34,35].On the one hand DCs can be generated ex vivo from monocytes and loaded with different leukemia associated antigens and on the other hand DC/DC leu can be generated directly from leukemic blasts presenting patients' individual antigen repertoire [9,10,36].Re-administration of these ex vivo generated and prepared DC/DC leu have shown to increase the frequencies of leukemia-specific (T) cells in vivo and the treatment achieved stable complete remissions in elderly AML patients [37][38][39].A new and interesting approach could be to induce the production of DC/DC leu from leukemic blasts in vivo after the treatment of patients with DC/DC leu -inducing response modifiers [6].

Hypoxia, a Condition with Influence on Haematological and Immune Reactions
Hematopoietic and immunoreactive cells are exposed to changing O 2 concentrations in the BM of about 0.1-1%, in arterial blood of about 12% and in PB of about 4-15% [20,21].Several groups have utilized hypoxic conditions at 6% O 2 to simulate physiological conditions ex vivo [21,40].Up to now, no consistent effect of hypoxia on the immune system was shown [41].Some studies suggest that hypoxia might suppress the anti-leukemic effect of immune cells, weakening the success of current anti-leukemic therapies [42].Moreover a correlation between the hypoxic tumor microenvironment and poor response to radiation/chemotherapy in patients was seen [42].Furthermore, physiological hypoxia could stimulate the proliferation and activation of NK cells, contributing to anti-leukemic functions [23].Different studies have demonstrated that the effects of oxygen on immune cells depend on the tissue and the duration of hypoxia exposure.For example, while 6% O 2 is considered as hypoxic, this oxygen concentration represents a normal physiological condition in the BM and stem cell niche [43,44].Therefore, we used 10% O 2 as an average of venous and arterial blood, as described previously [20,21].Moreover, intense blast proliferation and O 2 consumption in leukemic-PB might affect the biology of leukemic blasts [21,45] compared to standard normoxic condition.In hypoxic conditions, the expression of membrane receptors (e.g., CXCR4) and the activation of intracellular signaling cascades of pO 2 -sensitive tumor suppressor genes (e.g., MAPK, HIF1a) change [40].Physiological hypoxia was shown to induce cell cycle arrest in the G0/G1-phase of AML blasts (cell lines and primary AML samples) by increasing the expression of the anti-apoptotic XIAP and activation of PI3K/Akt [46].

Generation of DC/DC leu from Leukemic Cell Lines
We utilized AML cell lines in the initial phase to assess the impact of hypoxia, as previous studies have demonstrated that AML cell lines serve as valuable tools for investigating the effects of hypoxia on leukemic cell proliferation [47].Our findings indicate that DCs can be successfully generated using DC/DC leu -generating protocols, irrespective of the type and the mutation status of the cell lines, under both hypoxic and normoxic conditions.Interestingly, our results revealed no significant decrease in the frequencies of generated DC/DC leu and no discernible differences in the blast proliferation between the two conditions.The adaptation to low oxygen levels during DC/DC leu -cultures can be attributed to the activity of HIF-1α [45].Notably, we observed higher frequencies of cells in the S-phase under normoxic conditions compared to hypoxic conditions in our experiments which might result in a lower cell proliferation activity in hypoxic compared to normoxic conditions.In summary, our study confirmed that DC/DC leu -generation from leukemic cell lines is feasible under hypoxic conditions, yielding comparable outcomes to those obtained under normoxic conditions.

DC/DC leu -Generation from Healthy and AML PBMNCs and WB
The generation of DC/DC leu using DC/DC leu -generating protocols from healthy and AML PBMNCs with Pici -PGE1 and Pici -PGE2 was successful compared to controls.These data confirm previous findings described by others and us [6,9,27,30].Interestingly, all results obtained were similar under both hypoxic and normoxic conditions.These results demonstrate that hypoxia, as a physiological condition, is not necessary for the ex vivo generation of DC/DC leu for later on adoptive cell transfer.
Whole blood contains all soluble and cellular factors present in the individual AML patient, which may have activating or inhibitory influences on physiological immune reactions [19].Therefore, we generated DC/DC leu directly from healthy and leukemic WB to simulate most physiological conditions.DC/DC leu generation was feasible under both hypoxic and normoxic conditions and the DC leu subgroups did not significantly differ between the two conditions.Importantly, the used Kits did not induce blasts' proliferation during cultures, thereby conforming previous findings.In the clinical context, these results support the idea that AML patients could be treated directly with Kits inducing DC/DC leugeneration in vivo [48,49].

DC/DC leu -Stimulation in T Cell Enriched MLC Results in Activated Cells of the Innate and Adaptive Immune System
T, iNKT, NK and CIK cells and their subsets are important mediators of innate and adaptive immune responses and their anti-tumor and anti-infections functionality are known to be activated by DC/DC leu [15].This was already confirmed previously under normoxic conditions using DC/DC leu generated with Kits in T cell enriched MLC [9,14,15,30,50].We can add that this activation was seen under hypoxic as well as normoxic conditions and resulted in comparable frequencies of different immune cell subsets.Memory T cells play a critical role in the maintenance of the complete remission of AML patients if activated in vivo [51,52].Furthermore, cells of the innate immunity significantly increased after MLC under hypoxic and normoxic conditions.

DC/DC leu Stimulation after T Cell Enriched MLC Results in Improved Anti-Leukemic Activity
As already shown before treatment of WB with Kits, followed by T cell enriched MLC improved anti-leukemic reactivity under normoxic conditions [25,50].Here we show that anti-leukemic activity was improved under hypoxic vs normoxic conditions.These results might point to different killing mechanisms under hypoxic vs normoxic conditions or at least a variation of this process (e.g., slow pathway of Fas/FasL-or the fast pathway of perforin-granzyme-mediated killing).These effects might act synergistically or independently [53,54].Additionally, the superior anti-leukemic activity observed after MLC WB-DC Kit I and MLC WB-DC Kit M appeared to be equally effective under normoxic conditions.However, a significantly higher number of cases with increased lysis after MLC WB-DC Kit M compared to controls (MLC WB ) was found under hypoxic versus normoxic conditions.These results suggest that hypoxia, as a physiological condition may enhance the anti-leukemic activity.

Correlation of Anti-Leukemic Cytotoxicity of Immunoreactive Cells Stimulated by DC/DC leu
We investigated the correlation between the anti-leukemic reactivity of effector cells (T cell-enriched WB treated with or without Kits) and the frequencies of various DC and immune cell subtypes.In normoxic conditions, a significant positive correlation was found between DC + /WB generated with Kit M and the highest anti-leukemic activity after 3 or 24 h of treatment with MLC WB-DC Kit M .Under hypoxic conditions, a significant correlation was observed between DC + /WB and DC leu /WB generated with Kit M and the most effective anti-leukemic reactivity of T effector cells after MLC WB-DC Kit M after 3 or 24 h.Other cell subtypes did not show a direct correlation with achieved cytotoxicity.These findings support previous studies indicating a relationship between DC stimulation and the gained cytotoxicity of stimulated T cells against leukemia [55].Moreover, the better anti-leukemic reactivity of MLC WB-DC Kit M compared to MLC WB could be a good explanation for the positive correlation between (DC leu /WB) generated with Kit M and the best anti-leukemic activity of T cells (MLC WB-DC Kit M ) in hypoxic conditions.

Conclusions
DC/DC leu -generation with Kit I and Kit M was shown to increase DC/DC leu frequencies and to activate different immune cells after T cell enriched MLC under hypoxic and normoxic conditions in comparable frequencies using AML cell lines, PBMNCs and WB from leukemic and healthy samples.Induced anti-leukemic reactions were shown to be superior under hypoxic vs normoxic conditions.In summary, we show that immunoreactions induced by DC/DC leu might be underestimated under normoxic conditions-pointing to improved effects of DC/DC leu -immunotherapies in vivo in the hypoxic niches of the body.However, additional studies are needed to evaluate the impact of hypoxic conditions on the immune system and the generation of DC/DC leu in vivo.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Figure 1 .
Figure 1.Characterization of AML cell lines and the generation of DC/DCleu under hypoxic and normoxic conditions.qPCR Real Time Quantitative Polymerase chain reaction; AML acute myeloid leukemia; mRNA messenger RNA; hypoxic conditions (5% O2 saturation); normoxic conditions (21% O2 saturation); CT cycle threshold; GAPDH glyceraldehyde-3-phosphate dehydrogenase gene, DCmat mature DC; DC dendritic cells; DCleu dendritic cells of leukemic origin; Bla leukemic blast, hypoxic conditions (5% O2 saturation); normoxic conditions (21% O2 saturation); S synthesis phase of the cell cycles; G1 gap/growth 1 phase of the cell cycles; G2 gap/growth 2 phase of the cell cycle.* p-values between 0.1 and 0.05, ** p-values between 0.05 and 0.005, *** p-values < 0.005, **** p-values < 0.0005.(A).qPCR analyses of fusion genes Four different AML cell lines (NB-4, Mono-Mac-6, KG-1 and THP-1) were cultured in the RPMI medium under hypoxic as well as normoxic conditions.Results show no differences between the expression of fusion genes (PML-RARA fusion gene in the NB-4 cell line, FGFR1OP2-FGFR1 fusion gene in the KG-1 cell lines, KMT2A-MLLT3 fusion gene in the Mono-Mac-6 cell line and KMT2A-MLLT3 fusion gene in the THP-1 cell line) under hypoxic (left side) or normoxic (right side) conditions.Relative expressions of fusion genes specific for each cell line were tested after five passages.The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as the housekeeping control.The y-axis shows the ΔΔCT differences between the genes of interest and the housekeeping gene.Average frequencies ± standard deviation are given.(B).DC/DCleu subsets after DC/DCleu-generation Results of DC/DCleu subsets after DC/DCleugeneration from four different cell lines lines (NB-4, Mono-Mac-6, KG-1, and THP-1) with Kit I, Kit

Figure 1 .
Figure 1.Characterization of AML cell lines and the generation of DC/DC leu under hypoxic and normoxic conditions.qPCR Real Time Quantitative Polymerase chain reaction; AML acute myeloid leukemia; mRNA messenger RNA; hypoxic conditions (5% O 2 saturation); normoxic conditions (21% O 2 saturation); CT cycle threshold; GAPDH glyceraldehyde-3-phosphate dehydrogenase gene, DC mat mature DC; DC dendritic cells; DC leu dendritic cells of leukemic origin; Bla leukemic blast, hypoxic conditions (5% O 2 saturation); normoxic conditions (21% O 2 saturation); S synthesis phase of the cell cycles; G1 gap/growth 1 phase of the cell cycles; G2 gap/growth 2 phase of the cell cycle.* p-values between 0.1 and 0.05, ** p-values between 0.05 and 0.005, *** p-values < 0.005, **** p-values < 0.0005.(A).qPCR analyses of fusion genes Four different AML cell lines (NB-4, Mono-Mac-6, KG-1 and THP-1) were cultured in the RPMI medium under hypoxic as well as normoxic conditions.Results show no differences between the expression of fusion genes (PML-RARA fusion gene in the NB-4 cell line, FGFR1OP2-FGFR1 fusion gene in the KG-1 cell lines, KMT2A-MLLT3 fusion gene in the Mono-Mac-6 cell line and KMT2A-MLLT3 fusion gene in the THP-1 cell line) under hypoxic (left side) or normoxic (right side) conditions.Relative expressions of fusion genes specific for each cell line were tested after five passages.The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as the housekeeping control.The y-axis shows the ∆∆CT differences between the genes of interest and the housekeeping gene.Average frequencies ± standard deviation are given.(B).DC/DC leu subsets after DC/DC leu -generation Results of DC/DC leu subsets after DC/DC leugeneration from four different cell lines lines (NB-4, Mono-Mac-6, KG-1, and THP-1) with Kit I, Kit M and control (culture without added response modifiers) are given.Only in the NB-4 cell line could significantly higher frequencies of mature DCs be generated with Kit I and Kit M under hypoxic compared to normoxic conditions.For all other subsets, comparable frequencies could be generated.Average frequencies ± standard deviations are given.(C).Cell cycle analysis Cells were fixed in ethanol, stained with propidium iodide (PI) and analysed for DNA content to determine populations in G1 and S phases of the cell cycle.Significantly lower frequencies of cells are found in the Sphase under hypoxic vs. normoxic conditions for the NB-4, KG-1 and THP-1 cell lines.Average frequencies ± standard deviation are given.

Figure 3 .
Figure 3. DC/DCleu generation from leukemic WB under hypoxic and normoxic conditions.WB whole blood; DC dendritic cells; hypoxic conditions (10% O2 saturation); normoxic conditions (21% O2 saturation), DC dendritic cells; DCleu dendritic cells of leukemic origin; DCmat mature dendritic cells.(A).Leukemic WB samples were cultured in parallel under hypoxic and normoxic conditions with Kit I and Kit M compared to controls (without added response modifiers).Proportions of cases with 'sufficient DC-generation' (setting a cut-off-value at 5% DC + /WB) from leukemic WB were not different under hypoxic (left side) and normoxic conditions (right sight).Each dot (• ▼ ) characterizes DC-frequencies generated from each AML-patient in each given case.(B).Leukemic WB samples were cultured with Kit I, Kit M and without added response modifiers (control) under hypoxic (left side) and normoxic (right sight) conditions.Average frequencies ± standard deviation of DCs and their subtypes are given.* p-values between 0.1 and 0.05, ** p-values between 0.05 and 0.005, *** p-values < 0.005.

Figure 3 .
Figure 3. DC/DC leu generation from leukemic WB under hypoxic and normoxic conditions.WB whole blood; DC dendritic cells; hypoxic conditions (10% O 2 saturation); normoxic conditions (21% O 2 saturation), DC dendritic cells; DC leu dendritic cells of leukemic origin; DC mat mature dendritic cells.(A).Leukemic WB samples were cultured in parallel under hypoxic and normoxic conditions with Kit I and Kit M compared to controls (without added response modifiers).Proportions of cases with 'sufficient DC-generation' (setting a cut-off-value at 5% DC + /WB) from leukemic WB were not different under hypoxic (left side) and normoxic conditions (right sight).Each dot (• ▼) characterizes DC-frequencies generated from each AML-patient in each given case.(B).Leukemic WB samples were cultured with Kit I, Kit M and without added response modifiers (control) under hypoxic (left side) and normoxic (right sight) conditions.Average frequencies ± standard deviation of DCs and their subtypes are given.* p-values between 0.1 and 0.05, ** p-values between 0.05 and 0.005, *** p-values < 0.005.

Figure 4 .
Figure 4. Composition of immunoreactive cells after T cell enriched MLC under hypoxic and normoxic conditions.% percentage; (A) shows amount of cells of the adaptive immune system, (B) shows amounts of cells of the innate immune system.MLC mixed lymphocyte cultures; MLC WB-DC Kit I and MLC WB-DC Kit M MLC with Kit I or Kit M pretreated WB, MLC WB control; hypoxic conditions (10% O2 saturation); normoxic conditions (21% O2 saturation).* p-values between 0.1 and 0.05, ** pvalues between 0.05 and 0.005, *** p-values < 0.005.

Figure 4 .
Figure 4. Composition of immunoreactive cells after T cell enriched MLC under hypoxic and normoxic conditions.% percentage; (A) shows amount of cells of the adaptive immune system, (B) shows amounts of cells of the innate immune system.MLC mixed lymphocyte cultures; MLC WB-DC Kit I and MLC WB-DC Kit M MLC with Kit I or Kit M pretreated WB, MLC WB control; hypoxic conditions (10% O 2 saturation); normoxic conditions (21% O 2 saturation).* p-values between 0.1 and 0.05, ** p-values between 0.05 and 0.005, *** p-values < 0.005.

Cancers 2024 , 21 Figure 5 .
Figure 5. Lysis of leukemic blasts after T cell enriched MLC under hypoxic and normoxic conditions.% percentage; h hours; MLC T cell enriched mixed lymphocyte cultures; MLC WB-DC Kit I and MLC WB- DC Kit M MLC with Kit I or Kit M pretreated WB; MLC WB control.** p-values between 0.05 and 0.005.

Figure 5 .
Figure 5. Lysis of leukemic blasts after T cell enriched MLC under hypoxic and normoxic conditions.% percentage; h hours; MLC T cell enriched mixed lymphocyte cultures; MLC WB-DC Kit I and MLC WB-DC Kit M MLC with Kit I or Kit M pretreated WB; MLC WB control.** p-values between 0.05 and 0.005.

Figure 6 .
Figure 6.Correlation analyses of cases with improved anti-leukemic reactivity and DC and DCleu under hypoxic (A,C) and normoxic conditions (B,D).DC dendritic cells; WB whole blood; DCleu dendritic cells of leukemic origin.Improved anti-leukemic activity after MLC WB-DC Kit M in comparison to MLC WB (y axis) correlated positively with frequencies of generated DC + /WB and DCleu/WB with Kit M (x axis) under hypoxic (left) and normoxic (right) conditions.Correlation was evaluated with pearson correlation analyses.

Figure 6 .
Figure 6.Correlation analyses of cases with improved anti-leukemic reactivity and DC and DC leu under hypoxic (A,C) and normoxic conditions (B,D).DC dendritic cells; WB whole blood; DC leu dendritic cells of leukemic origin.Improved anti-leukemic activity after MLC WB-DC Kit M in comparison to MLC WB (y axis) correlated positively with frequencies of generated DC + /WB and DC leu /WB with Kit M (x axis) under hypoxic (left) and normoxic (right) conditions.Correlation was evaluated with pearson correlation analyses.
. DC/DC leu -Based Immunotherapy for AML

Author
Contributions: F.D.-G.conducted cell-culture experiments under hypoxic conditions for cell lines, PBMNCs and W.B. experiments, analysed the corresponding data, conducted cell-lineculture experiments also under normoxic conditions.D.C.A. conducted cell-culture experiments for PBMNCs and W.B. under normoxic conditions and rafted this manuscript together with H.M.S., C.A., M.W., C.S. (Christoph Schwepcke), L.K., O.S. supported F.D.-G. with DC/DC leu -culture-, MLCand CTX-experiments under normoxic and hypoxic conditions.H.H. drafted the figures used in this manuscript.D.K., A.R., C.S. (Christoph Schmid), provided patient samples.H.M.S. designed the study and was responsible for the funding.All authors have read and agreed to the published version of the manuscript.Funding: The project was supported by intramural funding from the working group of H.M.S. F.D.-G. was funded by grants from the DAAD (ID 21520154 and scholarship 2016-2017 of the Ludwig Maximilian University of Munich).The funders did not influence the study design, data collection or analysis, the publishing decision or the manuscript preparation.Institutional Review Board Statement: Samples were collected and patients' informed consent gathered according to the Helsinki guidelines and the vote of the Ethics Committee of the Ludwig Maximilian University of Munich (vote number: 339-05).

Table 1 .
Characteristics of AML Patients and AML Cell Lines.
Pat. # Patient's number; f female; m male; CD cluster of differentiation; s secondary AML; p primary AML; bold blasts markers used to detect DC leu ; n.d.no data; * evaluated by flow cytometry; PB peripheral blood.

Table 3 .
Monocytes, leukemic blasts, DC/DC leu and T cell substes as evulated by flow cytometry.