Generation of Leukaemia-Derived Dendritic Cells (DCleu) to Improve Anti-Leukaemic Activity in AML: Selection of the Most Efficient Response Modifier Combinations

Dendritic cells (DC) and leukaemia derived DC (DCleu) are potent stimulators of anti-leukaemic activity in acute myeloid leukaemia (AML) and can be generated from mononuclear cells in vitro following standard DC/DCleu-generating protocols. With respect to future clinical applications though, DC/DCleu-generating protocols specifically designed for application in a whole-blood-(WB)-environment must be established. Therefore, we developed ten new DC/DCleu-generating protocols (kits; Kit-A/-C/-D/-E/-F/-G/-H/-I/-K/-M) for the generation of DC/DCleu from leukaemic WB, containing calcium-ionophore, granulocyte-macrophage-colony-stimulating-factor (GM-CSF), tumour-necrosis-factor-alpha, prostaglandin-E1 (PGE1), prostaglandin-E2 (PGE2) and/or picibanil (OK-432). All protocols were evaluated regarding their performance in generating DC/DCleu using refined classification and/or ranking systems; DC/DCleu were evaluated regarding their performance in stimulating anti-leukaemic activity using a cytotoxicity fluorolysis assay. Overall, we found the new kits capable to generate (mature) DC/DCleu from leukaemic WB. Through refined classification and ranking systems, we were able to select Kit-I (GM-CSF + OK-432), -K (GM-CSF + PGE2) and -M (GM-CSF + PGE1) as the most efficient kits in generating (mature) DC/DCleu, which are further competent to stimulate immunoreactive cells to show an improved anti-leukaemic cytotoxicity as well. This great performance of Kit-I, -K and -M in mediating DC/DCleu-based anti-leukaemic immunity in a WB-environment in vitro constitutes an important and directive step for translating DC/DCleu-based immunotherapy of AML into clinical application.


Introduction
Acute myeloid leukaemia is a malignant disorder of the hematopoietic system, characterised by an uncontrolled proliferation of abnormally differentiated myeloid blasts [1,2]. It is the predominant subtype of leukaemia in adults with a median age of 72 years at diagnosis [3]. Standard treatment options include chemotherapy with or without allogeneic stem cell transplantation (SCT) [4,5], which have an overall 5-year-survival-rate of

DC/DCleu-Generation Using Standard Protocols Is Comparable from MNC and WB
We were able to generate significant frequencies of (mature) DC and DCleu from both MNC and WB with standard methods (Ca, Mcm, Pici). Pooling all standard methods, we found no significant differences in frequencies of DC/cells, DCleu/cells and DCleu/DC comparing MNC-and WB-cultures, but significantly higher frequencies of DCmig/DC in WB- With regard to future clinical applications though, more physiological conditions have to be endeavoured, and thus, progression from DC/DC leu -generation in an MNC-to a whole-blood-(WB)-environment is inevitable. WB, in contrary to MNC, not only contains the full spectrum of soluble (e.g., cytokines, chemokines) and cellular (e.g., granulocytes, erythrocytes) components that take part in an immune response but also that determine the patient-and tumour-specific environment, consequently influencing the generation of DC/DC leu and the mediation of DC/DC leu -based anti-leukaemic immunity.
In this study, we developed DC/DC leu -generating protocols specifically designed for the generation of DC/DC leu in a WB-environment and evaluated their potential to generate DC/DC leu and mediate DC/DC leu -based anti-leukaemic immunity. This constitutes an important and directive step for translating DC/DC leu -based immunotherapy into clinical application.

DC/DC leu -Generation Using Standard Protocols Is Comparable from MNC and WB
We were able to generate significant frequencies of (mature) DC and DC leu from both MNC and WB with standard methods (Ca, Mcm, Pici). Pooling all standard methods, we found no significant differences in frequencies of DC/cells, DC leu /cells and DC leu /DC comparing MNC-and WB-cultures, but significantly higher frequencies of DC mig /DC in WB-cultures compared to MNC-cultures ( Figure 2A). Considering all standard methods separately, we found no significant differences in frequencies of DC/cells, DC leu /cells and DC leu /DC comparing MNC-and WB-cultures but (significantly) higher frequencies of DC mig /DC in WB-cultures compared to MNC-cultures ( Figure 2B, exemplary shown Pici; Figure A1).
Evaluating proportions of cases with sufficient and insufficient DC/DC leu -generation according to the DC/DC leu -classification (Table 1), we found higher proportions of cases with sufficient DC/DC leu -generation using WB compared to MNC after treatment with all three standard protocols ( Figure 2C). In summary, we found the generation of (mature) DC/DC leu using standard protocols equivalent or even superior in WB compared to MNC. Subsequent experiments were thus performed in a WB-environment simulating more physiological conditions.

DC/DC leu -Generation from WB Is Comparable Using New Protocols (Kits) and Standard Protocols
We introduced nine new DC/DC leu -generating protocols (kits; Kit-A, -C, -D, -E, -F, -G, -H, -I, -K) and compared their capacity to generate DC and DC leu (subgroups) to the performance of the standard protocols they derived from in a WB-environment. We thus compared results of Kit-A, -C, -E, -G, -K to Mcm (sharing GM-CSF, TNFa and/or PGE 2 ), Kit-E, -H to Mcm (all cytokine-based), Kit-D, -G, -I to Pici (sharing GM-CSF and/or OK-432) and Kit-F to Ca (sharing Ca-Iono).
We found no significant differences in frequencies of DC and DC leu (subtypes) generated with Kit-A, -C, -D, -G, -I, -K compared to standard protocols ( Figure 3A, exemplarily shown Kit-I vs. Pici; Figure A2). Kit-F even generated significantly higher frequencies of DC/WB and DC leu /WB compared to Ca ( Figure 3B). However, Kit-E, -H generated significantly lower frequencies of DC/WB and DC leu /WB compared to Mcm ( Figure 3C, exemplarily shown Kit-E vs. Mcm; Figure A2). Frequencies of DC leu /DC and DC mig /DC were comparable between kits and standard protocols.

Protocols
We introduced nine new DC/DCleu-generating protocols (kits; Kit-A, -C, -D, -E, -F, -G, -H, -I, -K) and compared their capacity to generate DC and DCleu (subgroups) to the performance of the standard protocols they derived from in a WB-environment. We thus compared results of Kit-A, -C, -E, -G, -K to Mcm (sharing GM-CSF, TNFa and/or PGE2), Kit-E, -H to Mcm (all cytokine-based), Kit-D, -G, -I to Pici (sharing GM-CSF and/or OK-432) and Kit-F to Ca (sharing Ca-Iono).
We found no significant differences in frequencies of DC and DCleu (subtypes) generated with Kit-A, -C, -D, -G, -I, -K compared to standard protocols ( Figure 3A, exemplarily shown Kit-I vs. Pici; Figure A2). Kit-F even generated significantly higher frequencies of DC/WB and DCleu/WB compared to Ca ( Figure 3B). However, Kit-E, -H generated significantly lower frequencies of DC/WB and DCleu/WB compared to Mcm ( Figure 3C, exemplarily shown Kit-E vs. Mcm; Figure A2). Frequencies of DCleu/DC and DCmig/DC were comparable between kits and standard protocols. Evaluating proportions of cases with sufficient (excellent, good, satisfactory) and insufficient DC/DCleu-generation according to the DC/DCleu-classification, we found Kit-F (compared to Ca), Kit-D and -I (compared to Pici) and Kit-A, -G and -K (compared to Mcm) generating higher proportions of cases with excellent DC/DCleu-generation compared to their respective standard protocol. Proportions of cases with good DC/DCleu-generation were lower with all kits, proportions of cases with satisfactory DC/DCleu-generation were lower with Kit-A, -D, -F, -I and equal or higher with Kit-C, -E, -G, -H, -K compared to their respective standard protocol ( Figure 4). Evaluating proportions of cases with sufficient (excellent, good, satisfactory) and insufficient DC/DC leu -generation according to the DC/DC leu -classification, we found Kit-F (compared to Ca), Kit-D and -I (compared to Pici) and Kit-A, -G and -K (compared to Mcm) generating higher proportions of cases with excellent DC/DC leu -generation compared to their respective standard protocol. Proportions of cases with good DC/DC leu -generation were lower with all kits, proportions of cases with satisfactory DC/DC leu -generation were lower with Kit-A, -D, -F, -I and equal or higher with Kit-C, -E, -G, -H, -K compared to their respective standard protocol ( Figure 4).
DCmig/DC generated with Pici compared to Kit-I (A), Ca compared to Kit-F (B) and Mcm compared to Kit-E (C). Statistically significant differences (p values < 0.05) (two-tailed t-test) are given.
Evaluating proportions of cases with sufficient (excellent, good, satisfactory) and insufficient DC/DCleu-generation according to the DC/DCleu-classification, we found Kit-F (compared to Ca), Kit-D and -I (compared to Pici) and Kit-A, -G and -K (compared to Mcm) generating higher proportions of cases with excellent DC/DCleu-generation compared to their respective standard protocol. Proportions of cases with good DC/DCleu-generation were lower with all kits, proportions of cases with satisfactory DC/DCleu-generation were lower with Kit-A, -D, -F, -I and equal or higher with Kit-C, -E, -G, -H, -K compared to their respective standard protocol ( Figure 4). In summary, most kits were able to generate comparable or, in the case of Kit-F, even higher frequencies of DC/DCleu (subgroups) and higher proportions of cases with excellent DC/DCleu-generation compared to their respective standard protocol. Of note, Kit-E and -H were not able to generate comparable frequencies of DC/DCleu (subgroups) but rather generated significantly lower frequencies compared to their standard protocol.

Ranking of Kits
We assessed the potential of kits based on two different ranking methods: 1) the classranking based on the proportions of excellent, good, satisfactory and insufficient results of DC/DCleu-generation using quantities of generated DC/WB and DCleu/WB, and 2) the best-ranking based on the proportions of best and second-best results of DC/DCleu-generation using quantities of generated DC/WB.

Class-Ranking of Kits
We ranked kits based on quantities of generated DC/WB and DCleu/WB. We therefore developed three subordinate rankings (ranking 1-3) and one superordinate ranking (ranking 4) ( Figure 5A, Table 2). Ranking 1 considers kits based on their proportion of In summary, most kits were able to generate comparable or, in the case of Kit-F, even higher frequencies of DC/DC leu (subgroups) and higher proportions of cases with excellent DC/DC leu -generation compared to their respective standard protocol. Of note, Kit-E and -H were not able to generate comparable frequencies of DC/DC leu (subgroups) but rather generated significantly lower frequencies compared to their standard protocol.

Ranking of Kits
We assessed the potential of kits based on two different ranking methods: (1) the class-ranking based on the proportions of excellent, good, satisfactory and insufficient results of DC/DC leu -generation using quantities of generated DC/WB and DC leu /WB, and (2) the best-ranking based on the proportions of best and second-best results of DC/DC leugeneration using quantities of generated DC/WB.

Class-Ranking of Kits
We ranked kits based on quantities of generated DC/WB and DC leu /WB. We therefore developed three subordinate rankings (ranking 1-3) and one superordinate ranking (ranking 4) ( Figure 5A, Table 2). Ranking 1 considers kits based on their proportion of cases with excellent DC/DC leu -generation. In doing so, Kit-I and -K performed the best (68% and 58% of cases, respectively), followed by Kit-F, -A, -D, -G, -C, -E and -H. Ranking 2 considers kits based on their proportion of cases with excellent and good DC/DC leu -generation. In doing so, Kit-K and -I performed the best (83% and 72% of cases, respectively), followed by Kit-A, -F, -D, -C, -G, -E and -H. Ranking 3 considers kits based on their proportion of cases with sufficient (excellent, good, satisfactory) DC/DC leu -generation. In doing so, Kit-K and -I performed the best (83% and 79% of cases, respectively), followed by Kit-A, -F, -D, -C, -G, -E and -H. Ranking 4 considers results of rankings 1-3, creating a superordinate ranking, nominating Kit-K and -I as the best performing kits, followed by Kit-A, -F, -D, -C, -G, -E and -H. In summary, the class-ranking of kits found Kit-K and -I to be the best performing kits, the best-ranking of kits found Kit-F, -A, -D and -I to be the best performing kits. Overall, Kit-I appears to be the most proficient kit in generating DC/DCleu.

Selection of Best Kits
We selected the best performing kits for further evaluation. Ranking of kits found Kit-K, -I, -A, -D and -F to be the most proficient kits. As TNFa and Ca-Iono were no longer approved for human systemic treatment though, we had to exclude Kit-A and -F from further considerations. As Kit-D, composed of three response modifiers (GM-CSF, OK-432, PGE2), showed no advantage in generating DC/DCleu compared to Kit-I and -K, composed of two response modifiers (GM-CSF with OK-432 or PGE2), we moreover excluded Given are (A) the classranking with the proportions of cases with sufficient (excellent, good, satisfactory) and insufficient DC/DC leu -generation using kits and (B) the best-ranking with proportions of cases with best-and second-best DC/DC leu -generation using kits. Table 2. Class-ranking of all kits.

Ranking 3 Excellent, Good, Satisfactory
were ranked based on the proportions of cases with excellent, good, and/or satisfactory generation of DC/WB and DC leu /WB.

Best-Ranking of Kits
We further ranked kits based on the best and second-best results of quantities of generated DC/WB. We therefore developed two subordinate rankings (ranking 1-2) and one superordinate ranking (ranking 3) ( Figure 5B, Table 3). Ranking 1 considers kits based on their proportion of cases with the best DC/DC leu -generation. In doing so, Kit-F and -A performed the best (33% and 30% of cases, respectively), followed by Kit-D, -I, -E, -C, -K, -G and -H. Ranking 2 considers kits based on their proportion of cases with the best and second-best DC/DC leu -generation. In doing so, Kit-F and -I performed the best (52% and 47% of cases, respectively), followed by Kit-D, -A, -E, -K, -C, -G and -H. Ranking 3 considers results of rankings 1-2, creating a superordinate ranking, nominating Kit-F and -A/-D/-I (the latter equally) as the best performing kits, followed by Kit-E, -K, -C, -G and -H. Table 3. Best-ranking of all kits.

Ranking 2 Best, Second-Best
were ranked based on the proportions of cases with best and/or second-best generation of DC/WB.
In summary, the class-ranking of kits found Kit-K and -I to be the best performing kits, the best-ranking of kits found Kit-F, -A, -D and -I to be the best performing kits. Overall, Kit-I appears to be the most proficient kit in generating DC/DC leu .

Selection of Best Kits
We selected the best performing kits for further evaluation. Ranking of kits found Kit-K, -I, -A, -D and -F to be the most proficient kits. As TNFa and Ca-Iono were no longer approved for human systemic treatment though, we had to exclude Kit-A and -F from further considerations. As Kit-D, composed of three response modifiers (GM-CSF, OK-432, PGE 2 ), showed no advantage in generating DC/DC leu compared to Kit-I and -K, composed of two response modifiers (GM-CSF with OK-432 or PGE 2 ), we moreover excluded Kit-D from further considerations. Lastly, as parallel studies with PGE 1 showed PGE 2 and PGE 1 to have comparable effects on the generation of DC/DC leu , we introduced a new Kit-M, derived from Kit-K, consisting of GM-CSF and PGE 1 , for further evaluation [37].
Overall, we selected Kit-I, -K and -M as the most promising kits and evaluated their capacity to generate (mature) DC/DC leu as well as their potential to initiate antileukaemic cytotoxicity.

Kit-I, -K and -M Generate Significantly Higher Frequencies of DC/DC leu Compared to Control
All three kits (Kit-I, -K, -M) were able to generate significantly higher frequencies of DC/WB and DC leu /WB compared to control ( Figure 6). Kit-generated DC/DC leu hereby consisted of significant frequencies of mature DC. Comparing kits among themselves, we found no significant differences in frequencies of generated DC/WB, DC leu /WB, DC leu /DC and DC mig /DC (data not shown). Ranking kits according to the class-ranking and bestranking, we found Kit-I to be the most efficient kit in generating DC/DC leu , followed by Kit-M and -K ( Figure 7A,B and Tables 4 and 5). Table 4 Class-ranking of selected kits.

Ranking 4 Combined
Selected kits (I, K, M) were ranked based on the proportions of cases with excellent, good and/or satisfactory generation of DC/WB and DCleu/WB. Table 5. Best-ranking of selected kits.

Ranking 3 Combined
Selected kits (I, K, M) were ranked based on the proportions of cases with best and/or second-best generation of DC/WB.      . Class-and best-ranking of Kit-I, -K and -M. Given are (A) the class-ranking with the proportions of cases with sufficient (excellent, good, satisfactory) and insufficient DC/DC leu -generation using kits and (B) the best-ranking with proportions of cases with best-and second-best DC/DC leugeneration using kits. Table 4. Class-ranking of selected kits.

Ranking 3 Excellent, Good, Satisfactory
Selected kits (I, K, M) were ranked based on the proportions of cases with excellent, good and/or satisfactory generation of DC/WB and DC leu /WB. Table 5. Best-ranking of selected kits.

Ranking 2 Best, Second-Best
Selected kits (I, K, M) were ranked based on the proportions of cases with best and/or second-best generation of DC/WB.

DC/DC leu Generated with Kit-I, -K and -M Stimulate Anti-Leukaemic Activity
We analysed the improvement in blast lytic activity of MLC Kit-I , MLC Kit-K and MLC Kit-M compared to MLC Control through CTX after 3 h and 24 h of incubation of effector and target cells to assess the anti-leukaemic activity of DC/DC leu -stimulated immunoreactive cells.
After 3 h, we could observe an improvement in blast lysis in about 40% of cases in MLC Kit-I , 60% of cases in MLC Kit-K and 50% of cases in MLC Kit-M compared to control. The average improvement in blast lysis was about 60% in MLC Kit-I , about 30% in MLC Kit-K and 50% in MLC Kit-M ( Figure 8A). After 24 h, we could observe an improvement in blast lysis in about 40% of cases in MLC Kit-I , 50% of cases in MLC Kit-K and 20% of cases in MLC Kit-M compared to control. The average improvement in blast lysis was about 75% in MLC Kit-I , about 20% in MLC Kit-K and 40% in MLC Kit-M ( Figure 8B). Selecting the best-achieved improvement in blast lysis after 3 h or 24 h, we could observe an improvement in blast lysis in about 60% of cases in MLC Kit-I , 80% of cases in MLC Kit-K and 70% of cases in MLC Kit-M compared to control. The average improvement in blast lysis was about 70% in MLC Kit-I , about 30% in MLC Kit-K and 50% in MLC Kit-M ( Figure 8C).  Figure 8A). After 24 h, we could observe an improvement in blast lysis in about 40% of cases in MLC Kit-I , 50% of cases in MLC Kit-K and 20% of cases in MLC Kit-M compared to control. The average improvement in blast lysis was about 75% in MLC Kit-I , about 20% in MLC Kit-K and 40% in MLC Kit-M ( Figure 8B). Selecting the best-achieved improvement in blast lysis after 3 h or 24 h, we could observe an improvement in blast lysis in about 60% of cases in MLC Kit-I , 80% of cases in MLC Kit-K and 70% of cases in MLC Kit-M compared to control. The average improvement in blast lysis was about 70% in MLC Kit-I , about 30% in MLC Kit-K and 50% in MLC Kit-M ( Figure 8C).  In summary, we found Kit-I, -K and -M gave rise to comparable frequencies of DC/DC leu . All three kits were able to improve the anti-leukaemic activity of immunoreactive cells compared to control. Overall, Kit-I, followed by Kit-M and -K, performed the best regarding the class-ranking and best-ranking, with the highest proportions of excellent and best DC/DC leu -generation, as well as the best-achieved improvement in blast lysis after 3 h or 24 h. We thus perceive Kit-I and -M to have the highest potential in generating DC/DC leu and stimulating anti-leukaemic activity.

DC leu -Based Immunotherapy
DC are one of the most potent mediators of the immune system. Manipulated to express leukaemia-specific antigens, they impose an auspicious way to (re-)activate the immune system to a leukaemia-specific immunity. Particularly, DC generated from myeloid leukaemic blasts (known as DC leu ), which are characterised by the simultaneous expression of dendritic-and leukaemia-specific antigens, hold the great potential of stimulating the immune system to the whole leukaemic antigen repertoire [15][16][17][18][19]. The process of converting myeloid leukaemic blasts into DC/DC leu in vitro using DC/DC leu -generating protocols consisting of various response modifiers has been demonstrated multiple times before [20,21,25]. Most protocols are based on an MNC-setting, though. With respect to future clinical applications, progression to DC/DC leu -generation in a WB-setting simulating a more physiological patient-and tumour-specific environment is inevitable. We thus developed new DC/DC leu -generating protocols specifically designed for the generation of DC/DC leu from leukaemic WB and evaluated their potential to generate DC/DC leu and mediate DC/DC leu -based anti-leukaemic activity.

Standard Protocols-DC/DC leu Generation from Leukaemic MNC and WB
Standard DC/DC leu -generating protocols like Ca, Mcm and Pici have been shown to be competent in generating significant frequencies of (mature) DC/DC leu from leukaemic MNC [20,21]. Although their compositions and operations were specifically designed for MNC-application, we nevertheless wanted to examine their performance in a novel WB-environment, simulating more physiological conditions. We found all standard protocols competent to generate DC/DC leu from MNC and WB to a comparable extent, with higher frequencies of mature DC generated from WB ( Figure 2). This is an interesting finding as the stimulation of maturation and CCR7-dependent (lymph node) migration is essential for DC/DC leu to activate T-cells as well as other immunoreactive cells [38][39][40]. DC maturation and migration is a tightly regulated process, controlled by a large variety of chemotactic and non-chemotactic factors like danger-associatedmolecular-patterns (DAMPs), acute-phase-proteins (APPs), proinflammatory cytokines or eicosanoids produced by different cell types [41]. A superior maturation of DC/DC leu in a WB-compared to an MNC-environment may be attributed to the fact that WB comprises the full spectrum of soluble (including chemotactic and non-chemotactic) and cellular factors necessary for the homeostasis of the blood and immune system and thus can readily support DC/DC leu maturation and migration. An inferior maturation of DC/DC leu in a (serum-free) MNC-environment in contrary suggests a restriction of this process due to the absence of (readily) available maturation and migration factors. This emphasises the importance of incorporating the full spectrum of soluble and cellular factors present in WB when developing immunomodulatory agents for in vivo use.
Assessing the overall DC/DC leu -generating potential of standard protocols using the DC/DC leu -classification, we found higher proportions of cases with sufficient DC/DC leugeneration using WB than MNC, letting DC/DC leu -generation with standard protocols appear more reliable from WB than MNC.
Overall, we found the standard protocols perform better in a WB-environment than in the MNC-environment they were originally designed for.

New Kits Specifically Designed for WB
We developed nine new DC/DC leu -generating protocols (kits) specifically designed for the generation of DC/DC leu from leukaemic WB. The new kits were derived from the standard protocols and composed of one to three response modifiers, including GM-CSF, IFNa, TNFa, PGE 1 , OK-432 and Ca-Iono.
We compared the performance of the new kits to their respective standard protocols and found Kit-F generated significantly higher, Kit-A, -C, -D, -G, -I and -K generated equal and Kit-E and -H generated significantly lower frequencies of DC/DC leu from WB compared to their respective standard protocol. Frequencies of mature DC were comparable between kits and standard protocols (Figure 3). Assessing kits using the DC/DC leu -classification showed a similar picture: we found Kit-F, -G (when compared to Mcm), -D, -I, -A and -K achieved higher proportions of cases with excellent DC/DC leugeneration and Kit-C, -E, -G (when compared to Pici) and -H achieved lower proportions of cases with excellent DC/DC leu -generation compared to their respective standard protocol ( Figure 4).
Overall, most kits performed comparable or even better compared to their respective standard protocol. Only Kit-E and -H performed worse in comparison.

Ranking of Kits
We further ranked kits according to their performance in generating DC/DC leu . Ranking kits with the class-ranking (based on proportions of cases with excellent, good and satisfactory generation of DC/DC leu ), we found Kit-K and -I achieved the best results ( Figure 5A, Table 2). Ranking kits with the best-ranking (based on proportions of cases with the best and second-best generation of DC), we found Kit-F and -A = -D = -I achieved the best results ( Figure 5B, Table 3).
Overall, we found Kit-K, -I, -A, -D and -F containing GM-CSF combined with OK-432, PGE 2 , TNFa and/or Ca-Iono the best performing kits, initiating sufficient DC/DC leugeneration in most cases. Interestingly, although GM-CSF has been shown to mediate differentiation, activation and maturation of dendritic cells [26,27], GM-CSF on its own, as in Kit-G, did not show sufficient DC/DC leu generation, which is in line with previous findings [42][43][44]. DC/DC leu -generation with GM-CSF seems to be dependent on the synergetic effects of additional response modifiers, like OK-432, PGE 2 or Ca-Iono, that further enhance dendritic differentiation and proliferation and moreover stimulate activation and maturation, properties essential for mediating a comprehensive immune response [42,45,46]. Of note, a combination of GM-CSF with two other response modifiers, OK-432 and PGE 2 , as in Kit-D, showed no advantage over a combination of GM-CSF with either OK-432 or PGE 2 , as in Kit-I and -K. Saturation of the DC/DC leu -generation-pathway might be at play here. The Ca-Iono containing Kit-F showed good results in line with previous findings [47,48]. Mentionable, Ca-Iono containing protocols like Kit-F hold the significant advantage of reduced culture times of two days without constrained success of DC/DC leugeneration and T-cell stimulation through generated DC/DC leu ; however, such protocols have constrained overall viability of DC/DC leu compared to other protocols [47,48]. When comparing the TNFa containing Kit-C to Kit-A, we found Kit-C performed worse than -A. This is dissenting with other findings that suggest PGE 2 to further enhance the effect GM-CSF together with TNFa [46,49]. The IFNa containing Kit-E and -H showed poor performance. This might be surprising at first as IFNa is well-known for its anti-tumour effects and its role in the treatment of several solid and haematological malignancies [50][51][52][53]. In AML, IFNa has been shown to directly inhibit blast proliferation and moreover stimulate blast apoptosis [51,54]-properties that yet come amiss when aiming to convert myeloid leukaemic blasts into DC leu , hampering the overall DC/DC leu -generating process.

Selection of Kits for (Potential) Treatment of Patients
In regard to future clinical applications, the development of kits has to consider not only the potential of kits but also their biocompatibility and applicability. Of the nine new kits we developed, Kit-K, -I, -A, -D and -F showed the best performance, thus qualifying for further in-depth evaluation. As TNFa and Ca-Iono were no longer approved for human systemic treatment, we had to exclude Kit-A and -F from further considerations. As Kit-D constitutes a combination of Kit-I and -K with no advantage to the individual protocols but with a more intricate composition of three response modifiers in contrast to two response modifiers of the individual protocols, we also excluded Kit-D from further considerations. Lastly, as parallel studies with PGE 1 showed PGE 2 and PGE 1 to have comparable effects on the generation of DC/DC leu , we introduced a new Kit-M, derived from Kit-K, consisting of GM-CSF and PGE 1 , for further evaluation [37].
Overall, we selected Kit-I, -K and -M as the most promising kits and further scrutinised their capacity to generate (mature) DC/DC leu as well as-and most importantly-their potential to initiate anti-leukaemic cytotoxicity through generated DC/DC leu . All three kits were able to generate significantly higher frequencies of DC/WB and DC leu /WB compared to the control ( Figure 6). Notably, Kit-generated DC/DC leu hereby consisted of significant frequencies of mature DC. Both class-and order-ranking determined Kit-I to be superior to Kit-M and -K ( Figure 7A,B and Tables 4 and 5). All three kits were able to improve the anti-leukaemic cytotoxicity of immunoreactive cells through generated DC/DC leu in most of the cases. Interestingly, some cases achieved an improvement in lysis after 3 h and some cases only after 24 h, whereas the average improvement in lysis was the highest in MLC Kit-K and MLC Kit-M after 3 h and in MLC Kit-I after 24 h. This can be explained by the different mechanisms by which immunoreactive cells exert cytotoxicity: the early and fast-acting perforin-granzyme pathway (as likely seen in MLC Kit-K and MLC Kit-M ) and the late and slow-acting Fas/FasL pathway (as likely seen in MLC Kit-I ), which can run separately or synergistically [55,56]. Overall, pooling the best anti-leukaemic cytotoxicity after 3 or 24 h, DC/DC leu generated with Kit-I appear to be the most efficient in stimulating blast-lytic activity, followed by Kit-M and -K (Figure 8).
Taken together, all three kits were able to generate significant amounts of (mature) DC/DC leu, which have the competence to regularly mediate anti-leukaemic cytotoxicity. Kit-I hereby performed the best, followed by Kit-M and -K, regarding the class-and best-ranking as well as the overall best-achieved improvement in blast lysis. We thus perceive Kit-I and -M to have the highest potential in generating DC/DC leu and stimulating anti-leukaemic activity.

Ongoing Kit Studies
Kit-I, -K, -M have shown auspicious results. On the way to successful in vivo treatment of patients with AML, kits though have to undergo further investigations regarding their performance and toxicity.
In this study, we could show that kits are competent to generate (mature) DC/DC leu that regularly initiate anti-leukaemic activity. We could confirm these results in multiple studies [24,37]. Furthermore, we could enlighten the leukaemia-specific activity of innate and adaptive immunoreactive cells on a single-cell level by analysing their IFNy secretion profiles and correlating them with the overall achieved anti-leukaemic cytotoxicity, thereby being able to display (indirectly and directly) participating immunoreactive cells of DC/DC leu -mediated leukaemia-specific and anti-leukaemic immune responses: levels of IFNy secreting innate (NK cells, CIK cells, iNKT cells) and adaptive (T-cells) immune cells were significantly higher in MLC Kits compared to MLC Control , with frequencies of IFNy secreting T CD3+ , T CD4+ , T CD8+ and NK 56+ cells positively correlating with the overall achieved anti-leukaemic activity [24]. When assessing compositions of DC/DC leu stimulated T-enriched immunoreactive cells, we found lower frequencies of regulatory T-cells (T reg ) in MLC Kits compared to MLC Control [57]; an important finding as the success of immunomodulation is directly affected by the T regs immunosuppressive disposition [58]. A milestone in drug development though not only is the verification of anti-leukaemic effects but also the exclusion of pro-leukaemic effects. We thus investigated the proliferation of blasts in relation to kit treatment but found no induction of blast proliferation through kit treatment in this regard [59]. Of note, DC/DC leu -generation and -functioning was comparable under normoxic (21% oxygen) and hypoxic (10% oxygen, resembling the in vivo situation) conditions [57].
Ultimately, DC/DC leu -based immunotherapy needs to be transferred from in vitro to in vivo settings to further assess its value in the treatment of AML. We thus extended our preclinical studies from in vitro to in vivo animal trials to gather more information about the kits' efficiency and toxicity. Kit treatment (Kit-M) of leukaemically diseased rats resulted in the generation of DC/DC leu without induction of blast proliferation and consecutively in an improved anti-leukaemic (blast-lytic) activity [60], displaying in vivo functionality of kits. For future studies, we are now focusing on conducting kit studies in patients with refractory AML, and striving to replicate the promising in vitro and in vivo results of DC/DC leu -based immunotherapy in clinic.

Sample Collection and Preparation
Samples were collected in the form of heparinised whole blood from patients in acute stages of AML and MDS after obtaining written consent and in accordance with the World Medical Association Declaration of Helsinki and the ethics committee of the Ludwig-Maximilian-University Hospital (vote no 33905). Samples were provided by the university hospitals of Munich, Tuebingen, Oldenburg and Augsburg.

Patients' Characteristics
Blood samples were obtained from patients in acute phases of AML (n = 65) and MDS (n = 2) with an average age of 54.3 (range 21-88) years and a female-to-male ratio of 1:1.3. Patients were classified based on the French-American-British (FAB) classification (M0-M5), the aetiology (primary AML, secondary AML), the stage of disease (diagnosis, persistence, relapse, relapse after SCT), the blast phenotype and frequency. An overview is given in Table 6.
Cell staining was performed by incubating cells with corresponding moAbs for 15 min in the dark using a staining medium containing 80% PBS (Biochrom) and 20% FCS (Biochrom). In the case of WB samples, erythrocytes were lysed prior to staining using a lysing buffer (Becton Dickinson) according to the manufacturers' instructions. Cells were analysed with the fluorescence-activated cell sorting flow cytometer FACS Calibur (Becton Dickinson) and the acquisition and analysis software CellQuestPro (Becton Dickinson). Isotype controls were conducted according to the manufacturer's instructions.

Mixed Lymphocyte Culture (MLC)
DC/DC leu from WB-cultures were used to stimulate T-cell enriched immunoreactive cells. Therefore, a fraction of DCC containing 25 × 10 3 DC/DC leu and 1 × 10 6 T-cells were co-cultured in 24-multiwell-culture-plates (Thermo Fisher Scientific) and diluted in 1 mL RMPI-1640 containing 100 U/mL penicillin. 50 U/mL interleukin-2 (IL-2) (PeproTech) were added to all cultures on day 0 and days 2-3. Cells were cultured following the protocols under standard conditions at 21% O 2 , 5% CO 2 and 37 • C. A half-medium exchange was carried out after 2-3 days.

Cytotoxicity Fluorolysis Assay (CTX)
The blast lytic activity of DC/DC leu stimulated T-cell enriched immunoreactive cells in MLC from WB-cultures was analysed using a cytotoxicity fluorolysis assay (CTX). Therefore, a fraction of MLC containing 1 × 10 6 T-cells (effector cells) was co-cultured with 1 × 10 6 thawed autologous leukaemic blasts (target cells) for 3 and 24 h at 37 • C, 21% O 2 and 5% CO 2 . Target cells were stained with blast specific moAbs before culture and with 7AAD and a particular number of fluorosphere beads (Beckman Coulter) after culture when harvested. As a control, effector and target cells were cultured under the same conditions but separated and only combined prior to flow cytometric analyses.
Flow cytometric analyses were performed using a refined gating strategy [21], whereas the achieved blast lytic activity was defined as the percentual difference of viable target cells (blasts) between the effector-target-co-culture and the control.

Statistical Methods
Data are presented as mean ± standard deviation (SD). Statistical analyses were implemented using the two-tailed t-test. Significance was defined as 'not significant' (n.s.) with p values > 0.10, as 'borderline significant' with p values 0.10 to 0.05, as 'significant' with p values 0.05 to 0.005 and as 'highly significant' with p values < 0.005. All statistical analyses and figures were implemented using Excel 2013 (Microsoft, Redmond, WA, USA), SPSS (IBM, Armonk, NY, USA) and Prism 9 (GraphPad Software, San Diego, CA, USA).

Conclusions
In this study, we developed DC/DC leu -generating protocols (kits; comprising one to three response modifiers) specifically designed for the application in a WB-environment. We assessed kits based on their capacity to generate mature DC/DC leu from leukaemic myeloid blasts and to mediate DC/DC leu -based anti-leukaemic immunity. Ultimately, we identified Kit-I, -K and -M as the best performing kits in vitro, qualifying for further evaluation in vitro and vivo. In regard to future clinical applications, these findings are of particular importance as they are the basis for establishing clinical protocols for successful DC/DC leu -based immunotherapy of AML patients in vivo.

Patents
Modiblast Pharma GmbH (Oberhaching, Germany) holds the European Patent 'EP 3,217,975 B1 and the US Patent 'US 10,912,820 , in which H.M.S. is involved.  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. Figure A1. DC/DCleu-generation from leukaemic WB and MNC with standard DC/DCleu-generating protocols (Ca, Mcm, Pici). Given are the mean ± SD of DC/cells, DCleu/cells, DCleu/DC and DCmig/DC generated with the standard protocol Ca (A) and Pici (B) from MNC compared to WB. Statistically significant differences (p values < 0.05) (two-tailed t-test) are given.