Different Immunotherapeutic Combinations Enhance Specific T Cell Immune Responses Against Leukemic Cells, as well as Leukemic Progenitor and Stem Cells, in Acute Myeloid Leukemia

Different Immunotherapeutic Combinations Enhance Specific T Cell Immune Responses Against Leukemic Cells, as well as Leukemic Progenitor and Stem Cells, in Acute Myeloid Leukemia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Different Immunotherapeutic Combinations Enhance Specific T Cell Immune Responses Against Leukemic Cells, as well as Leukemic Progenitor and Stem Cells, in Acute Myeloid Leukemia Jochen Greiner, Patrick Schuler, Hubert Schrezenmeier, Johanna Weiss, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6523399/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Oct, 2025 Read the published version in Leukemia → Version 1 posted 10 You are reading this latest preprint version Abstract Immunotherapeutic approaches have become increasingly important in cancer therapy, including for patients with acute myeloid leukaemia (AML). Despite being shown to be effective in the context of stem cell transplants for almost 50 years, further improvements are required to prevent relapse and its associated morbidity. The therapeutic use of immune checkpoint inhibition in AML is still under debate. We have shown some positive effects of it on cancer control ex vivo . We found that anti-programmed death-1 (PD-1) antibodies in combination with azacitidine (AZA) had the most pronounced effect on T-cell activation and control of leukemic progenitor/ stem cell growth. We identified which leukemia-associated antigen (LAA) stimulated the largest IFNg immune response by T cells from AML patients with and without the nucleophosmin 1 (NPM1) mutation and which immunotherapeutic strategy, either alone or in combination with anti-PD-1, could enhance immune responses against leukemic cells and leukemic progenitor/stem cells. Anti-PD-1 with AZA had a particularly strong effect with a mean colony reduction of 56% (range: 0-100%). Taken together, combinations of immunotherapeutic approaches increase antigen-specific immune responses against leukemic cells but also leukaemic progenitor/stem cells. Especially the combination of LAA-peptides with anti-PD-1 antibody and one further immunotherapeutic could be an interesting option for further clinical studies. Health sciences/Health care/Therapeutics/Immunotherapy Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia Biological sciences/Immunology/Cell death and immune response Biological sciences/Stem cells/Haematopoietic stem cells Health sciences/Medical research/Preclinical research acute myeloid leukaemia NPM1 immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Acute myeloid leukemia (AML) is a form of cancer that affects the bone marrow and is characterized by an abnormal growth and interruption in the differentiation of myeloid progenitor cells. AML primarily affects older individuals, a population that often lacks the physical strength for intensive treatment. While chemotherapy is the standard treatment for AML, alone it is insufficient at preventing the high rate of relapse among adults ( 1 ). The 5-year survival rate for patients with AML is still poor, underscoring the urgent demand for innovative and synergistic treatments ( 2 ). In recent years, there has been a growing emphasis on finding effective immunotherapy methods for AML, specifically those that target leukemia cells and their precursors. Given the complexity of AML, it may be beneficial to combine various immunotherapy approaches in a strategic manner to prevent escape mechanisms and limit potential toxicity ( 3 – 5 ). Immunotherapeutic options, which are now commonly used in AML treatment, such as allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion, have shown significant success but also carry risks ( 6 , 7 ). On the other hand, other immunotherapeutic approaches such as immunotherapies with vaccines, bi-/tri-specific antibodies, hypomethylating agents and CAR-T-cell treatment, to mention only a few, are still in the earlier stages of development and require further improvement ( 8 – 10 ). Targeted immunotherapy in cancer treatment has become ever more important in recent years. The efficacy of immunotherapeutic approaches such as immune-checkpoint inhibitors (ICI) ( 11 , 12 ), chimeric antigen receptor (CAR) T cells ( 13 ) or bi-specific T cell activating antibodies ( 14 ) are becoming increasingly apparent. Also, the role of epigenetic immunomodulating drugs (such as histone deacetylase inhibitors) in combination with the immune-checkpoint inhibitor anti-Programmed death-1 (anti-PD-1, Nivolumab®) is up for debate. However, mechanisms of these immune responses and responsible antigen structures have to be further elucidated. Since leukemia-associated antigens (LAA) are target structures relevant for the elimination of malignant cells by cytotoxic T lymphocytes (CTL), they represent immunogenic antigens that are candidates for specific immunotherapy. In fact, in immunotherapy, it is feasible to use combinations of different therapeutic options mostly because their targets differ ( 15 ). However, the impact of an immunomodulating drug and ICI to attenuate LAA-specific immune responses has not been studied until now. Especially in AML it could be of paramount importance to use specific target antigens ( 16 ). We have previously shown that anti-PD-1 enhanced the cytotoxic effects of LAA-stimulated CTL against leukemic progenitor and stem cells (LPC/LSCs). This was most pronounced against nucleophosmin 1 mutated (NPM1 mut ) AML cells when the immunogenic epitope was derived from the mutated region of NPM1 ( 17 ). In this study, we investigated whether different immunotherapeutics alone or in combination with anti-PD-1 can further enhance the LPC/LSC degradation of NPM1 mut AML in comparison to AML cells with a wild type NPM1 gene (NPM1 WT ). Immune checkpoint therapy may have the capacity to reverse immune evasion in myeloid malignancies. While monotherapy with ICIs has produced only modest results, the combination with hypomethylating agents, intensive chemotherapy and several immune checkpoint inhibitors (ICIs) have shown promising results ( 18 ). Additionally, these inhibitors act as positive regulators of the immune system and are often used as an initial treatment to improve response rates and increase relapse-free survival after chemotherapy and bone marrow transplantation ( 19 ). Recent research also suggests the potential benefit of immunotherapy in the treatment of AML, specifically targeting CD33, CD123 and CLL-1, as well as the use of ICIs such as anti-PD-1 and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in the presence or absence of conventional chemotherapy ( 20 ). Single anti-PD-1 monoclonal antibody infusions have shown limited effectiveness in AML ( 21 ). However, in conjunction with hypomethylating agents (HMAs), they represent encouraging treatment options for relapsed/refractory AML patients and for older patients as a first-line treatment ( 22 ). Phase I/II trials with anti-PD-1/PD-L1 antibodies in combination with HMAs have shown encouraging and durable response rates, however there were small patient numbers, the toxicity was quite high and there were no randomized studies ( 23 ). The aim of this research is to examine ex vivo the potential of using specific T cells with various immunotherapies, either alone or in conjunction with anti-PD-1, to achieve a greater reduction of LPC/LSC, to compare NPM1 mut AML to NPM1 WT AML cells and to find a feasible way to minimize relapse rates by the use of combination immunotherapies. Participants and Methods Sample Preparation, Isolation and Freezing Samples were received from ten AML NPM1 mut and ten AML NPM1 WT participants following informed consent and in accordance with the Declaration of Helsinki. The local ethics committee (No. 334/09 and No. 221/14) approved the study protocol. All patient samples consisted of more than 90% leukemic blasts. Healthy donor (HD) samples were obtained from the German Red Cross in Ulm. Peripheral blood mononuclear cells (PBMCs) from AML patients and HDs were separated via Ficoll (Pan Biotech, Aidenbach, Germany) density gradient centrifugation, cryopreserved and stored in liquid nitrogen prior to use. Selection of LAAs To measure the response of specific CTLs, mononuclear cells (MNCs) from AML NPM WT patients were stimulated first with the LAA that generated the largest response against Preferentially expressed antigen in melanoma (PRAME; ALYVDSLFFL) or Wilms Tumor 1 (WT1; RMFPNAPYL), as described in previous analyses ( 24 ). MNCs from NPM mut patients were stimulated with the mutation related peptide NPM1 (AIQDLCVAV). Only HLA-A2 positive patient samples and HDs were used, since all LAAs were HLA-A2-restricted. The cytomegalovirus (CMV [NLVPMVATV]) peptide served as positive control, data not shown. All peptides were purchased form Proimmune, Oxford, GB. Mixed Lymphocyte Peptide Cultures (MLPCs) PBMC samples from HD were thawed, counted and divided into two equal portions. One fraction served as a source of antigen-presenting cells (APCs), which were irradiated with 30 Gy and pulsed with the respective peptides for 1.5 hours at 37°C, while the other portion was not irradiated and used for the generation of effector T cells with and without the addition of immunotherapeutics. In this way, peptide-specific CD8 + allogeneic T cells were generated from HD samples, providing a source of effector (E) cells for further testing. In brief, the appropriate immunotherapeutics were added alone or in combination with the E fraction. Then APCs were mixed with the E cells in a 1:1 ratio. On the second day, IL-2 (2.5 ng/ml) and IL-7 (20 ng/ml) were added and the culture was incubated for eight or nine days and then used for functional testing. Addition of Immunotherapeutics to Cell Culture In line with the results of a former titration ( 25 ), 5 µg of the anti-PD-1 antibody (nivolumab) and/or 5 µg of anti-CTLA4 (Ipilimumab; BMS, Munich, Germany)/ all-trans retinoic acid (ATRA; Vesanoid, Roche, Basel, Switzerland) / azacitidine (AZA; Vidaza, Celgene, New Jersey, USA) or the immunomodulator lenalidomide (Len; Revlimid, Celgene, Uxbridge, UK) were added on day 0 to the MLPC containing the E fraction alone for one hour, then the irradiated peptide loaded APCs were added to stimulate the MNC fraction. In this way, the direct effects of each ICI, alone and in combination, on E cells were measured. CD8 + T cells were stimulated with CMV or the respective LAA without immunotherapeutics as controls. Enzyme-Linked-Immuno-Spot (ELISpot) Membrane flat-bottomed 96 well plates (Merck Millipore Ltd, Carrigtwohill, Ireland) were coated with a solid antibody phase (mAb 1-D1K, 1.5 µg per well). Subsequently, the blocked membranes were incubated with allogeneic pre-stimulated MNCs from MLPC and peptide pulsed blasts from leukemia patients used as APC at a ratio of 5:1. Cytokines bound to the solid antibody phase were visualised with specific antibodies coupled to biotin (mAb 7-B6-1-biotin, 0.1 µg per well), alkaline phosphatase and the corresponding substrate. The evaluation was carried out using an Immunospot ELISpot reader. IFNg ELISpot (Mabtech, Nacka Strand, Sweden) was performed according to the manufacturer’s instructions. Colony-Forming Immunoassays (CFIs) Allogeneic T cells from MLPCs were used as a source of E cells and the ratio of E:Target (T) was 10:1. Primary patient T cells were used as a source of E and T cells stimulated with no peptide served as a growth control. After a brief and gentle centrifugation, E and T were resuspended in Iscove's Modified Dulbecco's Medium (IMDM) containing 2% fetal calf serum (FCS) and added to a 3 mL of hematopoietic stem cells-colony-forming unit (HSC-CFU) complete media with erythropoietin (StemMACS, Miltenyi Biotech, Bergisch Gladbach, Germany) and incubated at 37°C for 4 h. The media was then aspirated using a syringe. A total of 1.1 mL medium was placed into each cell culture dish (Thermo Scientific, Waltham, MA, USA). Colonies were analysed after a 20-day incubation time; the difference between control and sample in percent was calculated and displayed. Statistical Analysis Statistical tests were performed using GraphPad PRISM v8. The program was also used to evaluate assays, for comprehensive analysis, for organizing data and for graphing. As a statistical analysis, we used the RM (repeated measure) one-way or REML (mixed-effects model) ANOVA test, * = p < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001. Results We used functional CFI to investigate the effect of the checkpoint-inhibitor anti-PD-1 (nivolumab) alone or in combination with one of four other immunotherapeutics (ipilimumab, Len, ATRA, AZA) to determine the response of allogeneic LAA-specific T cells against leukemic cells and LPC/LSC taken from AML patients. 20 AML patient samples, of which 10 were from NPM1 mut and 10 were from NPM1 WT individuals, were tested in functional CFI assays to determine the immunogenicity of LAA and anti-PD-1-stimulated allogeneic CD8 + T cells with or without the four other immunotherapeutics to determine their impact on LPC/LSCs (Fig. 1 ). PBMC of HD were stimulated with the LAA that they had showed the strongest response to in former CFI assays to test the above immunotherapeutics alone or in combination with anti-PD-1 ( 25 , 26 ). The reduction in colonies (%) were found to be most notable after the addition of anti-PD-1 (anti-PD-1; nivolumab) when using a single immunotherapeutic agent. 13 of the 20 patient samples analysed showed a significant reduction in LPC/LSC-derived colony numbers in the presence of anti-PD-1, with an average reduction of 25% (range 0–75%, p 0.0023). In the presence of ATRA or when using anti-PD-1 and ATRA, the reduction was 8% and 18% respectively (range 0–38% vs. 0–65%, both p = ns). The immune response following the addition of anti-CTLA4 was 26% (p 0.0014); with anti-PD-1 and anti-CTLA4: 22% (p = 0.0138). In the presence of AZA, we observed a significant reduction in the number of LPC/LSC-derived colonies in 12 of 20 patient samples of 34% (range 0-100%, p < 0.0001). With Len the reduction measured was: 34% (range 0–95%, p < 0.0001) in 12 of 20 patient samples and with anti-PD-1/Len in 15 of 20 patient samples with a mean reduction of 44% (range 0–84%, p < 0.0001). However, in the presence of anti-PD-1 and AZA, the decrease in the percentage of LPC/LSC-derived colonies was most pronounced at 56% (range 0-100%, p < 0.0001) in 17 of 20 patient samples. When considering only the responders, several reduction rates were notable: anti-PD-1 responders (39%), AZA (57%), anti-PD-1/AZA (66%), Len alone (57%) versus anti-PD-1/Len (59%) RM one-way ANOVA * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. When considering the AML patients by virtue of NPM mutation status, in the ten NPM1 mut patient samples, the response rates were most notable for anti-PD-1 and AZA (NPM1 vs NPM1 anti-PD-1 and AZA 64%; p < 0.0001) and even lower in anti-PD-1 and Len (NPM1 vs NPM1 anti-PD-1 and Len 46%; p = 0.0004) (Fig. 2 ). Overall 6/6/9 patients of ten NPM1 mut patients responded when treated with anti-PD-1 or AZA or the combination of anti-PD-1/AZA. The average reduction rate for anti-PD-1 or AZA or the combination of anti-PD-1/AZA was 18/40/64% for all and 30/66/72% for responders only. Anti-PD-1 alone (18%) showed an increase in colony destruction compared to ATRA (8%) or anti-PD-1/ATRA (14%). Using anti-CTLA4 or the combination of anti-PD-1/ anti-CTLA4 the reduction rate was 29/21%. Len alone and anti-PD-1/Len had significant responses of 29 and 46%. Stimulated cells with LAA only served as negative controls, and were set to 0% reduction, meaning no reduction. In Fig. 2 B NPM1 WT patient samples are shown. When treated with anti-PD-1 or AZA, or anti-PD-1/AZA in combination, 7/6/8 of ten NPM1 WT patients responded, respectively; the average reduction rate was 32/28/41% for all and 46/47/59% for responders only. ATRA/CTLA4 (or the respective combinations) showed a lower reduction rate than anti-PD-1 alone. Len and anti-PD-1/Len had a significant response of 39 and 42%. Stimulated cells with LAA only served as growth control. RM one-way ANOVA * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. To extend our understanding of the effects of peptide stimulation on the clearance of AML patient cells by the immune system, we incubated MNCs from NPM1 mut patient samples with the NPM1 mut peptide and showed that this alone was sufficient to cause a significant reduction in colony numbers by 34% (25 patients, p < 0.0001; Fig. 3 ). This is an extended analysis of samples we had analysed previously ( 25 , 26 ) and patient (Pt) cells alone served as a negative control. The addition of anti-PD-1 and/or AZA augmented the reduction of colonies significantly when patient samples were treated with the LAA specific for NPM1 mut . Significant reduction of colonies was observed with NPM1 and anti-PD-1 of 66% (25 pts, p < 0.0001), NPM1 and AZA of 79% (10 pts, p < 0.0001) and NPM1 and anti-PD-1/AZA of 86% (10 pts, p < 0.0001). R We investigated the production of IFNγ by CD8 + T cells from two NPM1 mut patients (Pt17/18) and two NPM1 WT patients (Pt19/20). All data were normalized to the respective LAA, which were for mutated patients NPM1 mut and for NPM WT patients PRAME peptide. ELISpot results were compared to single CFI results (Fig. 4 ). The augmented stimulation of the two NPM1 mut patients (pt 17/18) with anti-PD-1 was 2.0/1.7-fold, ATRA 2.1/0.7-fold, anti-PD-1 and ATRA 2.8/1.0-fold, AZA 2.6/1.1-fold, anti-PD-1 and AZA 3.3/1.6-fold, anti-CTLA4 2.7/2.0-fold (p 0.0036), anti-PD-1 and anti-CTLA4 4.1/2.8-fold, Len 5.4/3.3-fold (p 0.0004), or anti-PD-1 and Len 4.7/3.3-fold (p 0.0009). AZA, CTLA4 and Len and its combinations resulted in a higher stimulation than anti-PD-1 alone. NPM1 mut patient cells were used in the ELISpot and although the results from ELISpot could not always be compared to colony reduction rates, this was possible with some results. When T cells from Pt17 were incubated with AZA, anti-PD-1/AZA, Len or anti-PD-1/Len in ELISpots there was an increased stimulation of T cells (Fig. 4 A) and a reduction in colony numbers (Fig. 4 B). In Pt. 18 with anti-PD-1/AZA, Len and anti-PD-1/Len, showed similar results (Fig. 4 A and C ). Thus, the effect of stimulating NPM1 mut CTL in the presence of immunotherapeutics against LPC/LSC could be demonstrated. When CTL from NPM WT Pt 19/20 were stimulated with anti-PD-1 and compared to LAA (PRAME) alone there was a 1.3/1.4-fold change in IFNγ secretion (Fig. 4 D). The other immunotherapeutics alone or in combination with anti-PD-1 showed the following fold changes compared to LAA (PRAME) alone: ATRA 1.1/1.2-fold, anti-PD-1 and ATRA 1.2/1.8-fold, AZA 1.3/1.0-fold, anti-PD-1 and AZA 1.1/2.4-fold, anti-CTLA4 2.3/2.2-fold, anti-PD-1 and anti-CTLA4 1.8/2.7-fold, Len 1.9/2.5-fold, and anti-PD-1 and Len 2.6/5.3-fold. The stimulation was lower in the NPM1 WT than in the NPM mut patients. In both Pt 19/20 (NPM1 WT patients) a synergistic effect between anti-PD-1 in combination with AZA could not be seen clearly, and was only observed when a combination of anti-PD-1/Len were used. In some patient samples the ELISpot results and CFI results correlated but not in others. Discussion AML is a multifaceted disease with numerous subtypes and complex genetic abnormalities ( 27 ). While treatments have improved in recent years, with remission rates of around 80%, half of patients experience relapse due to the emergence of drug-resistant clones ( 28 ). Bone marrow transplants have been used to treat leukemia patients for around 70 years. Meanwhile, immunotherapy has advanced and now also includes peripheral blood stem cell transplants, donor leukocyte infusions, monoclonal antibodies, adoptive T and/or NK cell therapy, checkpoint blockade and leukemia vaccines ( 29 ). Increasingly, conventional and immunotherapeutic approaches are being used in combination, especially in patients with relapsed and/or refractory disease ( 30 ). In melanoma, combinations of checkpoint inhibitor therapies showed durable clinical benefits. Wolchok et al reported on the 6.5-year CheckMate 067 results and showed improved outcomes with nivolumab plus ipilimumab or nivolumab versus ipilimumab in patients with advanced melanoma and the benefit of combination over nivolumab monotherapy ( 31 ). In AML the results differ as checkpoint inhibitor monotherapies have shown some clinical effect and improved overall survival in high-risk AML patients not eligible for allogeneic hematopoietic stem cell transplantation ( 32 ), while early-phase clinical trials suggest that ICIs and HMAs are safe and more promising ( 33 ). Another difficulty is that HMAs stimulate the immune response against tumor antigens while ICIs are upregulated. In our CFI assays, especially when using NPM1 mut AML samples, the results were promising when we used the combination of the checkpoint inhibitor PD-1 and AZA. Indeed, we were able to demonstrate a significant reduction in LPC/LSCs in primary AML patient samples in the presence of the ICI and HMA combination. Earlier, monotherapy with ATRA was used for the treatment of acute promyelocytic leukemia (APL) with high response rates, but the duration of response was short. Later, the development of ATRA, chemotherapy and arsenic trioxide combinations made APL a highly curable malignancy ( 34 ). We were interested therefore, whether a therapy with ATRA might be feasible for other types of AML, especially for patients with NPM1 mut . NPM1 mutations may render AML patients susceptible to ATRA, raising the possibility that a strategy targeting mutant oncoproteins to selectively induce NPM1 mutated protein degradation, that involved ATRA, may be a feasible alternative to specific pharmacologic NPM1 inhibitors. This could also be an approach that brings about the selective degradation of the NPM1 mutant proteins ( 35 , 36 ). However, our CFI results give an indication that there were no higher responses to ATRA in patients with AML mut compared to AML NPM1 WT patients. The HMAs, AZA and Decitabine, are the standard therapy for higher-risk myelodysplastic syndromes and for patients with AML who are not eligible for intensive therapy. In addition to optimized use in the various stages of disease, most clinical progress with HMAs has been achieved through the development of pharmacokinetically improved second-generation agents and the search for synergistic drug combinations based on HMAs, which has so far been predominantly empirical ( 37 ). There are ongoing attempts to prevent resistance to HMAs by using checkpoint inhibitors to boost the immune response ( 38 ). In our CFI, especially for NPM1 mut AML the results were promising as to the combination of the checkpoint inhibitor PD-1 with AZA. In fact, we could show significant reduction of LPC/LSC in primary AML patient samples. The LAA NPM1 already showed a significant reduction of 34% while with a single ICI (NPM1 and anti-PD-1) the reduction was 66%, with an HMA (NPM1 and AZA) the reduction was 79% and with the combination of NPM1 and ICI and HMA (NPM1, anti-PD-1 and AZA) the reduction of colonies increased to 86%. The difficulty, however, is that while HMAs stimulate the immune response against tumor antigens, the inhibitory ICIs were also upregulated. In our CFI, especially for NPM1 mut AML samples, the results were promising as to the combination of the checkpoint inhibitor PD-1 in combination with AZA. We were able to demonstrate a significant reduction of LPC/LSC in primary AML NPM1 mut patient samples. CTLA4 as single-agent has shown modest clinical activity in both relapsed/refractory (R/R) AML and MDS. The low mutational burden of AML may be a possible explanation for the lack of activity of T-cell activating ICIs, especially CTLA-4 and PD-1 inhibitors ( 11 , 12 ). We saw a moderate but solid reduction in stem cell-like cells with the checkpoint inhibitors CTLA4 or PD-1 alone, and again we saw that the combination did not enhance these effects ( 11 , 25 ). The immunomodulatory drug Len has anti-inflammatory, anti-proliferative, pro-apoptotic and anti-angiogenic properties and in this way promotes anti-tumour immunity. The effects of Len differ from those of other compounds used to treat AML, making Len an interesting agent for use in AML and in combination with existing agents ( 15 ). The issue of addressing resistance mechanisms in targeted therapy is another crucial consideration. The adaptability of cancer cells is significant because of clonal evolution ( 39 ). In the development of AML, somatic mutations accumulate in hematopoietic stem/progenitor cells, resulting in uncontrolled growth ( 40 ). This ultimately leads to the development of resistance mechanisms, evasion of the immune system and continued proliferation of leukemia cells ( 41 ). Taken together, we have shown that combinations of immunotherapeutic approaches increase antigen-specific immune responses against leukemic cells but also LPC/LSC, especially the combination of LAA peptides with the anti-PD-1 antibody and one further immunomodulating drug, like AZA, could be an interesting option for further clinical trials and might open up interesting application possibilities. Abbreviations AML: acute myeloid leukemia; APC: Antigen Presenting Cells; APL: acute promyelocytic leukemia; ATRA: all-trans retinoic acid; AZA: Azacitidine; CFI: Colony-Forming Immunoassays; CMV: cytomegalovirus; CTL: cytotoxic T lymphocytes; CTLA4: cytotoxic T-lymphocyte-associated protein 4; ICI: immune checkpoint inhibitor; HD: healthy donor; HMA: hypomethylating agents; LAA; leukemia-associated antigen; Len: Lenalidomide; LPC/LSC: leukemic progenitor/stem cells; MPLC: Mixed Lymphocyte Peptide Cultures; MNC: Mononuclear cell; mut: mutated; NPM: nucleophosmin; PBMC: Peripheral blood mononuclear cells; PD-1: Programmed death-1; PRAME: Preferentially expressed antigen in melanoma; WT1: Wilms Tumor 1; WT: wild type. Declarations Acknowledgments The authors thank all patients for the donation of samples. Conflicts of Interest The authors declare no conflict of interest. Ethics Patient samples were taken following informed consent and in accordance with the Declaration of Helsinki. The local ethics committee (No. 334/09 and No. 221/14) approved the study protocol. All patient samples consisted of more than 90% leukemic blasts. Anonymized samples from HDs were obtained from the German Red Cross in Ulm, processed by ficollization ( Pan Biotech, Aidenbach, Germany) and cryopreserved. Authors contributions Conception and design, J.G.; resources, P.J.S. and H.S.; methodology and validation, M.G., C.B. and J.W.; analysis and interpretation of results, J.G., P.J.S, M.G. and B.G.; writing - original draft preparation, J.G. and M.G.; writing - review and editing, J.G., P.J.S., H.S. and B.G.; supervision, J.G. and B.G. All authors have read and agreed to the published version of the manuscript. Data Availability The data generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Dohner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Buchner T, et al. 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A Review of Efficacy and Safety of Checkpoint Inhibitor for the Treatment of Acute Myeloid Leukemia. Frontiers in pharmacology. 2019;10:609. Daver N, Boddu P, Garcia-Manero G, Yadav SS, Sharma P, Allison J, et al. Hypomethylating agents in combination with immune checkpoint inhibitors in acute myeloid leukemia and myelodysplastic syndromes. Leukemia. 2018;32(5):1094–105. Schneider V, Zhang L, Rojewski M, Fekete N, Schrezenmeier H, Erle A, et al. Leukemic progenitor cells are susceptible to targeting by stimulated cytotoxic T cells against immunogenic leukemia-associated antigens. Int J Cancer. 2015;137(9):2083–92. Greiner J, Gotz M, Hofmann S, Schrezenmeier H, Wiesneth M, Bullinger L, et al. Specific T-cell immune responses against colony-forming cells including leukemic progenitor cells of AML patients were increased by immune checkpoint inhibition. Cancer immunology, immunotherapy: CII. 2020;69(4):629–40. Greiner J, Goetz M, Schuler PJ, Bulach C, Hofmann S, Schrezenmeier H, et al. Enhanced stimulation of antigen-specific immune responses against nucleophosmin 1 mutated acute myeloid leukaemia by an anti-programmed death 1 antibody. Br J Haematol. 2022;198(5):866–74. Weinberg OK, Porwit A, Orazi A, Hasserjian RP, Foucar K, Duncavage EJ, et al. The International Consensus Classification of acute myeloid leukemia. Virchows Arch. 2023;482(1):27–37. Dohner H, Weisdorf DJ, Bloomfield CD. Acute Myeloid Leukemia. N Engl J Med. 2015;373(12):1136–52. Greiner J, Gotz M, Wais V. Increasing Role of Targeted Immunotherapies in the Treatment of AML. International journal of molecular sciences. 2022;23(6). Thol F, Dohner H, Ganser A. How I treat refractory and relapsed acute myeloid leukemia. Blood. 2024;143(1):11–20. Wolchok JD, Chiarion-Sileni V, Gonzalez R, Grob JJ, Rutkowski P, Lao CD, et al. Long-Term Outcomes With Nivolumab Plus Ipilimumab or Nivolumab Alone Versus Ipilimumab in Patients With Advanced Melanoma. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2022;40(2):127–37. Reville PK, Kantarjian HM, Ravandi F, Jabbour E, DiNardo CD, Daver N, et al. Nivolumab maintenance in high-risk acute myeloid leukemia patients: a single-arm, open-label, phase II study. Blood cancer journal. 2021;11(3):60. Mendez LM, Posey RR, Pandolfi PP. The Interplay Between the Genetic and Immune Landscapes of AML: Mechanisms and Implications for Risk Stratification and Therapy. Frontiers in oncology. 2019;9:1162. Yilmaz M, Kantarjian H, Ravandi F. Acute promyelocytic leukemia current treatment algorithms. Blood cancer journal. 2021;11(6):123. Grant S. ATRA and ATO team up against NPM1. Blood. 2015;125(22):3369–71. Ranieri R, Pianigiani G, Sciabolacci S, Perriello VM, Marra A, Cardinali V, et al. Current status and future perspectives in targeted therapy of NPM1-mutated AML. Leukemia. 2022;36(10):2351–67. Duchmann M, Itzykson R. 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Cite Share Download PDF Status: Published Journal Publication published 27 Oct, 2025 Read the published version in Leukemia → Version 1 posted Editorial decision: revise 10 Jul, 2025 Review # 1 received at journal 07 Jul, 2025 Review # 2 received at journal 22 Jun, 2025 Reviewer # 2 agreed at journal 16 Jun, 2025 Reviewer # 1 agreed at journal 16 Jun, 2025 Reviewers invited by journal 15 Jun, 2025 Editor assigned by journal 12 Jun, 2025 Submission checks completed at journal 12 Jun, 2025 First submitted to journal 11 Jun, 2025 Unknown event 25 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6523399","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":471553551,"identity":"82d67b65-2db0-4350-84d6-a2677bc1e23f","order_by":0,"name":"Jochen Greiner","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie2PsUrEQBCGJwQ2zZ62uSKXV5iwlXBvYjNBuGs8ENIoWCwIuSZ47eUtDnyAiwwkzaFY2oiQFxDSKEQxOW2EW7UU3A92i2G++WcALJY/StG9ETC4X6qO/kFRnwqCFH2Nvld6Yl38VgkP+ZpPTh+ml5VbI7RtvA7nd83ZMwQLgxLdTIiXm2SWs1DkpBhnQibDDYHKDTFRJpEHKc1WDIpf9VahoSaIV4VJ2X/iwRtNkb2mgHarHL30ytqghFICDzQRslQEole88iPFcAtK0TWXFOUsE3RSpbLyWBzoia+WppTMrRt5TuHebXXlQzsK5hdVfa/H42BhOB93jJLYfb5hrS5lxyjv0dhusVgs/5J3D3Zcd4W3tvkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1256-173X","institution":"Diakonie Hospital Stuttgart","correspondingAuthor":true,"prefix":"","firstName":"Jochen","middleName":"","lastName":"Greiner","suffix":""},{"id":471553552,"identity":"a8f03a44-f9d2-412b-b96d-2b1b9ccde081","order_by":1,"name":"Patrick Schuler","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Schuler","suffix":""},{"id":471553553,"identity":"d38dda23-c137-4013-8f8d-e661b45e6123","order_by":2,"name":"Hubert Schrezenmeier","email":"","orcid":"","institution":"University of Berlin","correspondingAuthor":false,"prefix":"","firstName":"Hubert","middleName":"","lastName":"Schrezenmeier","suffix":""},{"id":471553554,"identity":"461ef98d-3dc1-45fd-be3c-7b8dc72a7592","order_by":3,"name":"Johanna Weiss","email":"","orcid":"","institution":"University Hospital Ulm","correspondingAuthor":false,"prefix":"","firstName":"Johanna","middleName":"","lastName":"Weiss","suffix":""},{"id":471553555,"identity":"f1633d05-60ca-4527-8ec9-9d5fb6e15005","order_by":4,"name":"Christiane Bulach","email":"","orcid":"","institution":"University Hospital Ulm","correspondingAuthor":false,"prefix":"","firstName":"Christiane","middleName":"","lastName":"Bulach","suffix":""},{"id":471553556,"identity":"3ed403fa-df5a-40b7-bb45-56e46b34fa04","order_by":5,"name":"Marlies Götz","email":"","orcid":"","institution":"University of Ulm","correspondingAuthor":false,"prefix":"","firstName":"Marlies","middleName":"","lastName":"Götz","suffix":""},{"id":471553557,"identity":"25af6190-4ab2-4c5c-8f51-5ce9f77019cb","order_by":6,"name":"Barbara Guinn","email":"","orcid":"https://orcid.org/0000-0003-0639-4541","institution":"University of Hull","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Guinn","suffix":""}],"badges":[],"createdAt":"2025-04-24 19:45:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6523399/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6523399/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41375-025-02764-7","type":"published","date":"2025-10-27T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84869113,"identity":"9fb981f4-ce4c-4946-ae39-f2c6ce872a01","added_by":"auto","created_at":"2025-06-18 08:47:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119128,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional CFI assays were performed with AML patient samples. The peptides were NPM1mut peptides for the 10 AML NPM1\u003csup\u003emut\u003c/sup\u003e patients and PRAME or WT1 for the AML NPM1\u003csup\u003eWT\u003c/sup\u003e patients. The immunogenicity of LAA with and without anti-PD-1-stimulated allogeneic CD8+ T cells, with and without other immunotherapeutics such as ATRA, AZA, CTLA4 and lenalidominde (Len) on LPC/LSC was analyzed with primary patient material. The number of colonies for LAA without immunotherapeutics served as a control. The immune responses to the stimulated LAA-specific T cells increased after the addition of anti-PD-1 (anti-PD-1; nivolumab). 13 of the 20 patient samples analysed showed a significant reduction in colony numbers when treated with anti-PD-1, with an average reduction of 25% (range 0-75%, p 0.0023). With ATRA and anti-PD-1 and ATRA, the reduction was 8% vs. 18% (respectively, range 0-38% vs. 0-65%, both p = ns). With AZA, we observed a significant reduction in 12 of 20 patient samples of 34% (range 0-100%, p \u0026lt;0.0001) and with anti-PD-1 and AZA, the decrease in colonies was even higher at 56% (range 0-100%, p \u0026lt;0.0001) in 17 of 20 patient samples. The immune response by addition of anti-CTLA4 was 26% (p 0.0014); with anti-PD-1 and anti-CTLA4: 22% (p 0.0138). With Len the reduction measured was 34% (range 0-95%, p \u0026lt;0.0001) in 12 of 20 patient samples and with anti-PD-1 and \u0026nbsp;Len in 15 of 20 patient samples with a mean reduction of 44% (range 0-84%, p \u0026lt;0.0001). If only the responders were considered, the reduction rates are even higher for anti-PD-1: 39%, for AZA 57%, for anti-PD-1 and AZA 66%, for Len alone versus anti-PD-1 and Len 57% versus 59%. RM one-way ANOVA * = p\u0026lt;0.05, ** = p\u0026lt;0.01, *** = p\u0026lt;0.001, **** = p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6523399/v1/30fa4f04a8806962d6182019.png"},{"id":84870503,"identity":"03880b4e-6009-44dc-ade5-358c856c7bf7","added_by":"auto","created_at":"2025-06-18 08:55:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57858,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional CFI assays were performed with NPM1\u003csup\u003emut\u003c/sup\u003e and NPM1\u003csup\u003eWT\u003c/sup\u003e patient samples. \u003cstrong\u003e(A) \u003c/strong\u003eOf the\u003cstrong\u003e \u003c/strong\u003eten NPM1\u003csup\u003emut\u003c/sup\u003e patients who were tested with anti-PD-1, AZA or anti-PD-1 and AZA in combination, 6, 6 and 9 patients responded; the average reduction rate was 18/40/64 for all and 30/66/72% for responders only. ATRA or anti-PD-1 and ATRA showed a lower reduction rate than anti-PD-1 alone; with CTLA4/\u003cstrong\u003e \u003c/strong\u003eanti-PD-1 and CTLA4 the reduction rate was 29/21%. Len /\u003cstrong\u003e \u003c/strong\u003eanti-PD-1 and Len had a significant response of 29/46%. Stimulated cells with LAA only served as negative control. \u003cstrong\u003e(B) \u003c/strong\u003eWhen 10 NPM1\u003csup\u003eWT\u003c/sup\u003e patient samples were treated with anti-PD-1/AZA/anti-PD-1 and AZA in combination respectively 7/6/8 responded; the average reduction rate was 32/28/47 for all and 46/47/59 % for responders only. ATRA/CTLA4 or the respective combinations showed a lower reduction rate than anti-PD-1 alone; Len /\u003cstrong\u003e \u003c/strong\u003eanti-PD-1 and Len had a significant response of 39/42%. Stimulated cells with LAA only served as negative control. RM one-way ANOVA * = p\u0026lt;0.05, ** = p\u0026lt;0.01, *** = p\u0026lt;0.001, **** = p\u0026lt;0.0001. The reduction in the number colonies is shown as a percentage (%) for each figure.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6523399/v1/8f989a37e3e991cca55536f7.png"},{"id":84869115,"identity":"95cb4c97-31ba-41d8-8a01-e3f4a913d56f","added_by":"auto","created_at":"2025-06-18 08:47:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55231,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003emmune\u003cstrong\u003e \u003c/strong\u003eeffects of NPM1, anti-PD-1, AZA and the combination of anti-PD-1 and AZA on AML NPM\u003csup\u003emut\u003c/sup\u003e colony numbers. Reductions were all measured compared to colonies formed by the same untreated patient cells. There was a significant reduction in colonies following NPM1mut stimulation in 25 pts 34% (p\u0026lt;0.0001), for NPM1 and anti-PD-1 in 25 pts (compared to pt cells alone) 66% (p\u0026lt;0.0001), for NPM1 and AZA in 10 pts 79% (p\u0026lt;0.0001), and for NPM1 and AZA and anti-PD-1 in 10 pts 86% (p\u0026lt;0.0001). REML ANOVA * = p\u0026lt;0.05, ** = p\u0026lt;0.01, *** = p\u0026lt;0.001, **** = p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6523399/v1/9e2a4eadca76ffabad25c6e2.png"},{"id":84869116,"identity":"d6908b2c-f898-4e32-a77f-5819de19047d","added_by":"auto","created_at":"2025-06-18 08:47:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103577,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFNg production by CD8+ T cells from NPM1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003emut\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e or NPM\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eWT\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e patients in the presence of stimulatory LAA.\u003c/strong\u003e ELISpot results were compared to CFI results for four patients and data were normalized to the respective LAA, which were either mutated NPM1, or for the NPM\u003csup\u003eWT\u003c/sup\u003e patients, PRAME. \u003cstrong\u003e(A)\u003c/strong\u003e Augmented stimulation of the two NPM1mut patients (pt 17 and 18) with anti-PD-1 was 2.0/1.7-fold. The other immunotherapeutics alone or in combination with anti-PD-1 showed the following fold changes: AZA and CTLA4, and Len and its combinations resulted in a higher stimulation than anti-PD-1 alone. \u003cstrong\u003e(B-C) \u003c/strong\u003eSingle patient CFU of mutated patient cells which were used in the ELISpot. The ELISpot results are not comparable in all cases, but in the mutant patient 17 results were observed with AZA and anti-PD-1/AZA and Len and anti-PD-1/Len, in which increased stimulation of the T cells in the ELISpot led to a reduction in colonies. In Pt. 18 with anti-PD-1/AZA, Len and anti-PD-1/Len, similar results could be seen. Thus, the effect of stimulation of specific CLT plus immunotherapeutics against LPC/LSC could be demonstrated. \u003cstrong\u003e(D)\u003c/strong\u003e The stimulation of the two NPM1 Wildtype patients, each stimulated and normalized to PRAME was as follows. Pt 19/20 with anti-PD-1 stimulation compared to LAA was 1.3/1.4-fold. The other immunotherapeutics alone or in combination with anti-PD-1 showed the following fold changes. ATRA 1.1/1.2-fold, anti-PD-1 and ATRA 1.2/1.8-fold, AZA 1.3/1.0-fold, anti-PD-1 and AZA 1.1/2.4-fold, anti-CTLA4 2.3/2.2-fold, anti-PD-1 and anti-CTLA4 1.8/2.7-fold, Len 1.9/2.5-fold, and anti-PD-1 and Len 2.6/5.3-fold (p 0.0058). The stimulation was lower in the Wildtype than in the mutated patients.\u003cstrong\u003e (E-F) \u003c/strong\u003eSingle patient CFU of patient wildtype cells which were also used in the ELISpot. In some patient samples the ELISpot results and CFI results correlated but not in others. Like in both patients considering the results anti-PD-1/Len, where the increased stimulation of T cells shown in the ELISpot resulted in a decrease in number of colonies. Interestingly, in the wildtype patient results the synergistic effect of anti-PD-1 in combination with AZA could not be seen clearly only the combination of anti-PD-1/Len could be correlated. RM one-way ANOVA * = p\u0026lt;0.05, ** = p\u0026lt;0.01, *** = p\u0026lt;0.001, **** = p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6523399/v1/10ac6f7adf85961a8a999d2f.png"},{"id":94583717,"identity":"4b40eca6-5678-4d48-a154-91a80d3a3bc9","added_by":"auto","created_at":"2025-10-28 18:14:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":839087,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6523399/v1/b738d0a7-b9af-41a7-8a3f-0e3071b19059.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Different Immunotherapeutic Combinations Enhance Specific T Cell Immune Responses Against Leukemic Cells, as well as Leukemic Progenitor and Stem Cells, in Acute Myeloid Leukemia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myeloid leukemia (AML) is a form of cancer that affects the bone marrow and is characterized by an abnormal growth and interruption in the differentiation of myeloid progenitor cells. AML primarily affects older individuals, a population that often lacks the physical strength for intensive treatment. While chemotherapy is the standard treatment for AML, alone it is insufficient at preventing the high rate of relapse among adults (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The 5-year survival rate for patients with AML is still poor, underscoring the urgent demand for innovative and synergistic treatments (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In recent years, there has been a growing emphasis on finding effective immunotherapy methods for AML, specifically those that target leukemia cells and their precursors. Given the complexity of AML, it may be beneficial to combine various immunotherapy approaches in a strategic manner to prevent escape mechanisms and limit potential toxicity (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImmunotherapeutic options, which are now commonly used in AML treatment, such as allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion, have shown significant success but also carry risks (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). On the other hand, other immunotherapeutic approaches such as immunotherapies with vaccines, bi-/tri-specific antibodies, hypomethylating agents and CAR-T-cell treatment, to mention only a few, are still in the earlier stages of development and require further improvement (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTargeted immunotherapy in cancer treatment has become ever more important in recent years. The efficacy of immunotherapeutic approaches such as immune-checkpoint inhibitors (ICI) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), chimeric antigen receptor (CAR) T cells (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) or bi-specific T cell activating antibodies (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) are becoming increasingly apparent. Also, the role of epigenetic immunomodulating drugs (such as histone deacetylase inhibitors) in combination with the immune-checkpoint inhibitor anti-Programmed death-1 (anti-PD-1, Nivolumab\u0026reg;) is up for debate. However, mechanisms of these immune responses and responsible antigen structures have to be further elucidated. Since leukemia-associated antigens (LAA) are target structures relevant for the elimination of malignant cells by cytotoxic T lymphocytes (CTL), they represent immunogenic antigens that are candidates for specific immunotherapy.\u003c/p\u003e \u003cp\u003eIn fact, in immunotherapy, it is feasible to use combinations of different therapeutic options mostly because their targets differ (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). However, the impact of an immunomodulating drug and ICI to attenuate LAA-specific immune responses has not been studied until now. Especially in AML it could be of paramount importance to use specific target antigens (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). We have previously shown that anti-PD-1 enhanced the cytotoxic effects of LAA-stimulated CTL against leukemic progenitor and stem cells (LPC/LSCs). This was most pronounced against nucleophosmin 1 mutated (NPM1\u003csup\u003emut\u003c/sup\u003e) AML cells when the immunogenic epitope was derived from the mutated region of NPM1 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In this study, we investigated whether different immunotherapeutics alone or in combination with anti-PD-1 can further enhance the LPC/LSC degradation of NPM1\u003csup\u003emut\u003c/sup\u003e AML in comparison to AML cells with a wild type NPM1 gene (NPM1\u003csup\u003eWT\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eImmune checkpoint therapy may have the capacity to reverse immune evasion in myeloid malignancies. While monotherapy with ICIs has produced only modest results, the combination with hypomethylating agents, intensive chemotherapy and several immune checkpoint inhibitors (ICIs) have shown promising results (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Additionally, these inhibitors act as positive regulators of the immune system and are often used as an initial treatment to improve response rates and increase relapse-free survival after chemotherapy and bone marrow transplantation (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Recent research also suggests the potential benefit of immunotherapy in the treatment of AML, specifically targeting CD33, CD123 and CLL-1, as well as the use of ICIs such as anti-PD-1 and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) in the presence or absence of conventional chemotherapy (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Single anti-PD-1 monoclonal antibody infusions have shown limited effectiveness in AML (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, in conjunction with hypomethylating agents (HMAs), they represent encouraging treatment options for relapsed/refractory AML patients and for older patients as a first-line treatment (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Phase I/II trials with anti-PD-1/PD-L1 antibodies in combination with HMAs have shown encouraging and durable response rates, however there were small patient numbers, the toxicity was quite high and there were no randomized studies (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe aim of this research is to examine \u003cem\u003eex vivo\u003c/em\u003e the potential of using specific T cells with various immunotherapies, either alone or in conjunction with anti-PD-1, to achieve a greater reduction of LPC/LSC, to compare NPM1\u003csup\u003emut\u003c/sup\u003e AML to NPM1\u003csup\u003eWT\u003c/sup\u003e AML cells and to find a feasible way to minimize relapse rates by the use of combination immunotherapies.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Participants and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample Preparation, Isolation and Freezing\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSamples were received from ten AML NPM1\u003csup\u003emut\u003c/sup\u003e and ten AML NPM1\u003csup\u003eWT\u003c/sup\u003e participants following informed consent and in accordance with the Declaration of Helsinki. The local ethics committee (No. 334/09 and No. 221/14) approved the study protocol. All patient samples consisted of more than 90% leukemic blasts. Healthy donor (HD) samples were obtained from the German Red Cross in Ulm. Peripheral blood mononuclear cells (PBMCs) from AML patients and HDs were separated via Ficoll (Pan Biotech, Aidenbach, Germany) density gradient centrifugation, cryopreserved and stored in liquid nitrogen prior to use.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSelection of LAAs\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo measure the response of specific CTLs, mononuclear cells (MNCs) from AML NPM\u003csup\u003eWT\u003c/sup\u003e patients were stimulated first with the LAA that generated the largest response against Preferentially expressed antigen in melanoma (PRAME; ALYVDSLFFL) or Wilms Tumor 1 (WT1; RMFPNAPYL), as described in previous analyses (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). MNCs from NPM\u003csup\u003emut\u003c/sup\u003e patients were stimulated with the mutation related peptide NPM1 (AIQDLCVAV). Only HLA-A2 positive patient samples and HDs were used, since all LAAs were HLA-A2-restricted. The cytomegalovirus (CMV [NLVPMVATV]) peptide served as positive control, data not shown. All peptides were purchased form Proimmune, Oxford, GB.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eMixed Lymphocyte Peptide Cultures (MLPCs)\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePBMC samples from HD were thawed, counted and divided into two equal portions. One fraction served as a source of antigen-presenting cells (APCs), which were irradiated with 30 Gy and pulsed with the respective peptides for 1.5 hours at 37\u0026deg;C, while the other portion was not irradiated and used for the generation of effector T cells with and without the addition of immunotherapeutics. In this way, peptide-specific CD8\u003csup\u003e+\u003c/sup\u003e allogeneic T cells were generated from HD samples, providing a source of effector (E) cells for further testing. In brief, the appropriate immunotherapeutics were added alone or in combination with the E fraction. Then APCs were mixed with the E cells in a 1:1 ratio. On the second day, IL-2 (2.5 ng/ml) and IL-7 (20 ng/ml) were added and the culture was incubated for eight or nine days and then used for functional testing.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eAddition of Immunotherapeutics to Cell Culture\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn line with the results of a former titration (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), 5 \u0026micro;g of the anti-PD-1 antibody (nivolumab) and/or 5 \u0026micro;g of anti-CTLA4 (Ipilimumab; BMS, Munich, Germany)/ all-trans retinoic acid (ATRA; Vesanoid, Roche, Basel, Switzerland) / azacitidine (AZA; Vidaza, Celgene, New Jersey, USA) or the immunomodulator lenalidomide (Len; Revlimid, Celgene, Uxbridge, UK) were added on day 0 to the MLPC containing the E fraction alone for one hour, then the irradiated peptide loaded APCs were added to stimulate the MNC fraction. In this way, the direct effects of each ICI, alone and in combination, on E cells were measured. CD8\u0026thinsp;+\u0026thinsp;T cells were stimulated with CMV or the respective LAA without immunotherapeutics as controls.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eEnzyme-Linked-Immuno-Spot (ELISpot)\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMembrane flat-bottomed 96 well plates (Merck Millipore Ltd, Carrigtwohill, Ireland) were coated with a solid antibody phase (mAb 1-D1K, 1.5 \u0026micro;g per well). Subsequently, the blocked membranes were incubated with allogeneic pre-stimulated MNCs from MLPC and peptide pulsed blasts from leukemia patients used as APC at a ratio of 5:1. Cytokines bound to the solid antibody phase were visualised with specific antibodies coupled to biotin (mAb 7-B6-1-biotin, 0.1 \u0026micro;g per well), alkaline phosphatase and the corresponding substrate. The evaluation was carried out using an Immunospot ELISpot reader. IFNg ELISpot (Mabtech, Nacka Strand, Sweden) was performed according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eColony-Forming Immunoassays (CFIs)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAllogeneic T cells from MLPCs were used as a source of E cells and the ratio of E:Target (T) was 10:1. Primary patient T cells were used as a source of E and T cells stimulated with no peptide served as a growth control. After a brief and gentle centrifugation, E and T were resuspended in Iscove's Modified Dulbecco's Medium (IMDM) containing 2% fetal calf serum (FCS) and added to a 3 mL of hematopoietic stem cells-colony-forming unit (HSC-CFU) complete media with erythropoietin (StemMACS, Miltenyi Biotech, Bergisch Gladbach, Germany) and incubated at 37\u0026deg;C for 4 h. The media was then aspirated using a syringe. A total of 1.1 mL medium was placed into each cell culture dish (Thermo Scientific, Waltham, MA, USA). Colonies were analysed after a 20-day incubation time; the difference between control and sample in percent was calculated and displayed.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eStatistical tests were performed using GraphPad PRISM v8. The program was also used to evaluate assays, for comprehensive analysis, for organizing data and for graphing. As a statistical analysis, we used the RM (repeated measure) one-way or REML (mixed-effects model) ANOVA test, * = p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** = p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** = p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and **** = p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe used functional CFI to investigate the effect of the checkpoint-inhibitor anti-PD-1 (nivolumab) alone or in combination with one of four other immunotherapeutics (ipilimumab, Len, ATRA, AZA) to determine the response of allogeneic LAA-specific T cells against leukemic cells and LPC/LSC taken from AML patients. 20 AML patient samples, of which 10 were from NPM1\u003csup\u003emut\u003c/sup\u003e and 10 were from NPM1\u003csup\u003eWT\u003c/sup\u003e individuals, were tested in functional CFI assays to determine the immunogenicity of LAA and anti-PD-1-stimulated allogeneic CD8\u0026thinsp;+\u0026thinsp;T cells with or without the four other immunotherapeutics to determine their impact on LPC/LSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PBMC of HD were stimulated with the LAA that they had showed the strongest response to in former CFI assays to test the above immunotherapeutics alone or in combination with anti-PD-1 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe reduction in colonies (%) were found to be most notable after the addition of anti-PD-1 (anti-PD-1; nivolumab) when using a single immunotherapeutic agent. 13 of the 20 patient samples analysed showed a significant reduction in LPC/LSC-derived colony numbers in the presence of anti-PD-1, with an average reduction of 25% (range 0\u0026ndash;75%, p 0.0023). In the presence of ATRA or when using anti-PD-1 and ATRA, the reduction was 8% and 18% respectively (range 0\u0026ndash;38% vs. 0\u0026ndash;65%, both p\u0026thinsp;=\u0026thinsp;ns). The immune response following the addition of anti-CTLA4 was 26% (p 0.0014); with anti-PD-1 and anti-CTLA4: 22% (p\u0026thinsp;=\u0026thinsp;0.0138). In the presence of AZA, we observed a significant reduction in the number of LPC/LSC-derived colonies in 12 of 20 patient samples of 34% (range 0-100%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). With Len the reduction measured was: 34% (range 0\u0026ndash;95%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in 12 of 20 patient samples and with anti-PD-1/Len in 15 of 20 patient samples with a mean reduction of 44% (range 0\u0026ndash;84%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). However, in the presence of anti-PD-1 and AZA, the decrease in the percentage of LPC/LSC-derived colonies was most pronounced at 56% (range 0-100%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in 17 of 20 patient samples.\u003c/p\u003e \u003cp\u003eWhen considering only the responders, several reduction rates were notable: anti-PD-1 responders (39%), AZA (57%), anti-PD-1/AZA (66%), Len alone (57%) versus anti-PD-1/Len (59%) RM one-way ANOVA * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003cp\u003eWhen considering the AML patients by virtue of NPM mutation status, in the ten NPM1\u003csup\u003emut\u003c/sup\u003e patient samples, the response rates were most notable for anti-PD-1 and AZA (NPM1 vs NPM1 anti-PD-1 and AZA 64%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and even lower in anti-PD-1 and Len (NPM1 vs NPM1 anti-PD-1 and Len 46%; p\u0026thinsp;=\u0026thinsp;0.0004) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Overall 6/6/9 patients of ten NPM1\u003csup\u003emut\u003c/sup\u003e patients responded when treated with anti-PD-1 or AZA or the combination of anti-PD-1/AZA. The average reduction rate for anti-PD-1 or AZA or the combination of anti-PD-1/AZA was 18/40/64% for all and 30/66/72% for responders only. Anti-PD-1 alone (18%) showed an increase in colony destruction compared to ATRA (8%) or anti-PD-1/ATRA (14%). Using anti-CTLA4 or the combination of anti-PD-1/ anti-CTLA4 the reduction rate was 29/21%. Len alone and anti-PD-1/Len had significant responses of 29 and 46%. Stimulated cells with LAA only served as negative controls, and were set to 0% reduction, meaning no reduction. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB NPM1\u003csup\u003eWT\u003c/sup\u003e patient samples are shown. When treated with anti-PD-1 or AZA, or anti-PD-1/AZA in combination, 7/6/8 of ten NPM1\u003csup\u003eWT\u003c/sup\u003e patients responded, respectively; the average reduction rate was 32/28/41% for all and 46/47/59% for responders only. ATRA/CTLA4 (or the respective combinations) showed a lower reduction rate than anti-PD-1 alone. Len and anti-PD-1/Len had a significant response of 39 and 42%. Stimulated cells with LAA only served as growth control. RM one-way ANOVA * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo extend our understanding of the effects of peptide stimulation on the clearance of AML patient cells by the immune system, we incubated MNCs from NPM1\u003csup\u003emut\u003c/sup\u003e patient samples with the NPM1\u003csup\u003emut\u003c/sup\u003e peptide and showed that this alone was sufficient to cause a significant reduction in colony numbers by 34% (25 patients, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This is an extended analysis of samples we had analysed previously (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and patient (Pt) cells alone served as a negative control. The addition of anti-PD-1 and/or AZA augmented the reduction of colonies significantly when patient samples were treated with the LAA specific for NPM1\u003csup\u003emut\u003c/sup\u003e. Significant reduction of colonies was observed with NPM1 and anti-PD-1 of 66% (25 pts, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), NPM1 and AZA of 79% (10 pts, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and NPM1 and anti-PD-1/AZA of 86% (10 pts, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). R\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWe investigated the production of IFNγ by CD8\u0026thinsp;+\u0026thinsp;T cells from two NPM1\u003csup\u003emut\u003c/sup\u003e patients (Pt17/18) and two NPM1\u003csup\u003eWT\u003c/sup\u003e patients (Pt19/20). All data were normalized to the respective LAA, which were for mutated patients NPM1\u003csup\u003emut\u003c/sup\u003e and for NPM\u003csup\u003eWT\u003c/sup\u003e patients PRAME peptide. ELISpot results were compared to single CFI results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The augmented stimulation of the two NPM1\u003csup\u003emut\u003c/sup\u003e patients (pt 17/18) with anti-PD-1 was 2.0/1.7-fold, ATRA 2.1/0.7-fold, anti-PD-1 and ATRA 2.8/1.0-fold, AZA 2.6/1.1-fold, anti-PD-1 and AZA 3.3/1.6-fold, anti-CTLA4 2.7/2.0-fold (p 0.0036), anti-PD-1 and anti-CTLA4 4.1/2.8-fold, Len 5.4/3.3-fold (p 0.0004), or anti-PD-1 and Len 4.7/3.3-fold (p 0.0009). AZA, CTLA4 and Len and its combinations resulted in a higher stimulation than anti-PD-1 alone. NPM1\u003csup\u003emut\u003c/sup\u003e patient cells were used in the ELISpot and although the results from ELISpot could not always be compared to colony reduction rates, this was possible with some results. When T cells from Pt17 were incubated with AZA, anti-PD-1/AZA, Len or anti-PD-1/Len in ELISpots there was an increased stimulation of T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and a reduction in colony numbers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In Pt. 18 with anti-PD-1/AZA, Len and anti-PD-1/Len, showed similar results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003eand C\u003c/b\u003e). Thus, the effect of stimulating NPM1\u003csup\u003emut\u003c/sup\u003e CTL in the presence of immunotherapeutics against LPC/LSC could be demonstrated. When CTL from NPM\u003csup\u003eWT\u003c/sup\u003e Pt 19/20 were stimulated with anti-PD-1 and compared to LAA (PRAME) alone there was a 1.3/1.4-fold change in IFNγ secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The other immunotherapeutics alone or in combination with anti-PD-1 showed the following fold changes compared to LAA (PRAME) alone: ATRA 1.1/1.2-fold, anti-PD-1 and ATRA 1.2/1.8-fold, AZA 1.3/1.0-fold, anti-PD-1 and AZA 1.1/2.4-fold, anti-CTLA4 2.3/2.2-fold, anti-PD-1 and anti-CTLA4 1.8/2.7-fold, Len 1.9/2.5-fold, and anti-PD-1 and Len 2.6/5.3-fold. The stimulation was lower in the NPM1\u003csup\u003eWT\u003c/sup\u003e than in the NPM\u003csup\u003emut\u003c/sup\u003e patients. In both Pt 19/20 (NPM1\u003csup\u003eWT\u003c/sup\u003e patients) a synergistic effect between anti-PD-1 in combination with AZA could not be seen clearly, and was only observed when a combination of anti-PD-1/Len were used. In some patient samples the ELISpot results and CFI results correlated but not in others.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAML is a multifaceted disease with numerous subtypes and complex genetic abnormalities (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). While treatments have improved in recent years, with remission rates of around 80%, half of patients experience relapse due to the emergence of drug-resistant clones (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBone marrow transplants have been used to treat leukemia patients for around 70 years. Meanwhile, immunotherapy has advanced and now also includes peripheral blood stem cell transplants, donor leukocyte infusions, monoclonal antibodies, adoptive T and/or NK cell therapy, checkpoint blockade and leukemia vaccines (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Increasingly, conventional and immunotherapeutic approaches are being used in combination, especially in patients with relapsed and/or refractory disease (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn melanoma, combinations of checkpoint inhibitor therapies showed durable clinical benefits. Wolchok et al reported on the 6.5-year CheckMate 067 results and showed improved outcomes with nivolumab plus ipilimumab or nivolumab versus ipilimumab in patients with advanced melanoma and the benefit of combination over nivolumab monotherapy (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In AML the results differ as checkpoint inhibitor monotherapies have shown some clinical effect and improved overall survival in high-risk AML patients not eligible for allogeneic hematopoietic stem cell transplantation (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), while early-phase clinical trials suggest that ICIs and HMAs are safe and more promising (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Another difficulty is that HMAs stimulate the immune response against tumor antigens while ICIs are upregulated. In our CFI assays, especially when using NPM1\u003csup\u003emut\u003c/sup\u003e AML samples, the results were promising when we used the combination of the checkpoint inhibitor PD-1 and AZA. Indeed, we were able to demonstrate a significant reduction in LPC/LSCs in primary AML patient samples in the presence of the ICI and HMA combination.\u003c/p\u003e \u003cp\u003eEarlier, monotherapy with ATRA was used for the treatment of acute promyelocytic leukemia (APL) with high response rates, but the duration of response was short. Later, the development of ATRA, chemotherapy and arsenic trioxide combinations made APL a highly curable malignancy (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). We were interested therefore, whether a therapy with ATRA might be feasible for other types of AML, especially for patients with NPM1\u003csup\u003emut\u003c/sup\u003e. NPM1 mutations may render AML patients susceptible to ATRA, raising the possibility that a strategy targeting mutant oncoproteins to selectively induce NPM1 mutated protein degradation, that involved ATRA, may be a feasible alternative to specific pharmacologic NPM1 inhibitors. This could also be an approach that brings about the selective degradation of the NPM1 mutant proteins (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). However, our CFI results give an indication that there were no higher responses to ATRA in patients with AML\u003csup\u003emut\u003c/sup\u003e compared to AML NPM1\u003csup\u003eWT\u003c/sup\u003e patients.\u003c/p\u003e \u003cp\u003eThe HMAs, AZA and Decitabine, are the standard therapy for higher-risk myelodysplastic syndromes and for patients with AML who are not eligible for intensive therapy. In addition to optimized use in the various stages of disease, most clinical progress with HMAs has been achieved through the development of pharmacokinetically improved second-generation agents and the search for synergistic drug combinations based on HMAs, which has so far been predominantly empirical (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). There are ongoing attempts to prevent resistance to HMAs by using checkpoint inhibitors to boost the immune response (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). In our CFI, especially for NPM1\u003csup\u003emut\u003c/sup\u003e AML the results were promising as to the combination of the checkpoint inhibitor PD-1 with AZA. In fact, we could show significant reduction of LPC/LSC in primary AML patient samples. The LAA NPM1 already showed a significant reduction of 34% while with a single ICI (NPM1 and anti-PD-1) the reduction was 66%, with an HMA (NPM1 and AZA) the reduction was 79% and with the combination of NPM1 and ICI and HMA (NPM1, anti-PD-1 and AZA) the reduction of colonies increased to 86%.\u003c/p\u003e \u003cp\u003eThe difficulty, however, is that while HMAs stimulate the immune response against tumor antigens, the inhibitory ICIs were also upregulated. In our CFI, especially for NPM1\u003csup\u003emut\u003c/sup\u003e AML samples, the results were promising as to the combination of the checkpoint inhibitor PD-1 in combination with AZA. We were able to demonstrate a significant reduction of LPC/LSC in primary AML NPM1\u003csup\u003emut\u003c/sup\u003e patient samples.\u003c/p\u003e \u003cp\u003eCTLA4 as single-agent has shown modest clinical activity in both relapsed/refractory (R/R) AML and MDS. The low mutational burden of AML may be a possible explanation for the lack of activity of T-cell activating ICIs, especially CTLA-4 and PD-1 inhibitors (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). We saw a moderate but solid reduction in stem cell-like cells with the checkpoint inhibitors CTLA4 or PD-1 alone, and again we saw that the combination did not enhance these effects (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe immunomodulatory drug Len has anti-inflammatory, anti-proliferative, pro-apoptotic and anti-angiogenic properties and in this way promotes anti-tumour immunity. The effects of Len differ from those of other compounds used to treat AML, making Len an interesting agent for use in AML and in combination with existing agents (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe issue of addressing resistance mechanisms in targeted therapy is another crucial consideration. The adaptability of cancer cells is significant because of clonal evolution (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). In the development of AML, somatic mutations accumulate in hematopoietic stem/progenitor cells, resulting in uncontrolled growth (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). This ultimately leads to the development of resistance mechanisms, evasion of the immune system and continued proliferation of leukemia cells (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, we have shown that combinations of immunotherapeutic approaches increase antigen-specific immune responses against leukemic cells but also LPC/LSC, especially the combination of LAA peptides with the anti-PD-1 antibody and one further immunomodulating drug, like AZA, could be an interesting option for further clinical trials and might open up interesting application possibilities.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAML: acute myeloid leukemia; APC: Antigen Presenting Cells; APL: acute promyelocytic leukemia; ATRA: all-trans retinoic acid; AZA: Azacitidine; CFI: Colony-Forming Immunoassays; CMV: cytomegalovirus; CTL: cytotoxic T lymphocytes; CTLA4: cytotoxic T-lymphocyte-associated protein 4; ICI: immune checkpoint inhibitor; HD: healthy donor; \u0026nbsp; HMA: hypomethylating agents; LAA; leukemia-associated antigen; Len: Lenalidomide; LPC/LSC: leukemic progenitor/stem cells; MPLC: Mixed Lymphocyte Peptide Cultures; MNC: Mononuclear cell; mut: mutated; NPM: nucleophosmin; PBMC: Peripheral blood mononuclear cells; PD-1: Programmed death-1; PRAME: Preferentially expressed antigen in melanoma; WT1: Wilms Tumor 1; WT: wild type.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank all patients for the donation of samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatient samples were taken following informed consent and in accordance with the Declaration of Helsinki. The local ethics committee (No. 334/09 and No. 221/14) approved the study protocol. All patient samples consisted of more than 90% leukemic blasts. Anonymized samples from HDs were obtained from the German Red Cross in Ulm, processed by \u003cem\u003eficollization (\u003c/em\u003ePan Biotech, Aidenbach, Germany) and cryopreserved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design, J.G.; resources, P.J.S. and H.S.; methodology and validation, M.G., C.B. and J.W.; analysis and interpretation of results, J.G., P.J.S, M.G. and B.G.; writing - original draft preparation, J.G. and M.G.; writing - review and editing, J.G., P.J.S., H.S. and B.G.; supervision, J.G. and B.G. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDohner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Buchner T, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKantarjian HM, Kadia TM, DiNardo CD, Welch MA, Ravandi F. Acute myeloid leukemia: Treatment and research outlook for 2021 and the MD Anderson approach. 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Nature reviews Clinical oncology. 2021;18(9):577\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"acute myeloid leukaemia, NPM1, immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-6523399/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6523399/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImmunotherapeutic approaches have become increasingly important in cancer therapy, including for patients with acute myeloid leukaemia (AML). Despite being shown to be effective in the context of stem cell transplants for almost 50 years, further improvements are required to prevent relapse and its associated morbidity.\u003c/p\u003e\n\u003cp\u003eThe therapeutic use of immune checkpoint inhibition in AML is still under debate. We have shown some positive effects of it on cancer control \u003cem\u003eex vivo\u003c/em\u003e. We found that anti-programmed death-1 (PD-1) antibodies in combination with azacitidine (AZA) had the most pronounced effect on T-cell activation and control of leukemic progenitor/ stem cell growth.\u003c/p\u003e\n\u003cp\u003eWe identified which leukemia-associated antigen (LAA) stimulated the largest IFNg immune response by T cells from AML patients with and without the nucleophosmin 1 (NPM1) mutation and which immunotherapeutic strategy, either alone or in combination with anti-PD-1, could enhance immune responses against leukemic cells and leukemic progenitor/stem cells. Anti-PD-1 with AZA had a particularly strong effect with a mean colony reduction of 56% (range: 0-100%).\u003c/p\u003e\n\u003cp\u003eTaken together, combinations of immunotherapeutic approaches increase antigen-specific immune responses against leukemic cells but also leukaemic progenitor/stem cells. \u0026nbsp;Especially the combination of LAA-peptides with anti-PD-1 antibody and one further immunotherapeutic could be an interesting option for further clinical studies.\u003c/p\u003e","manuscriptTitle":"Different Immunotherapeutic Combinations Enhance Specific T Cell Immune Responses Against Leukemic Cells, as well as Leukemic Progenitor and Stem Cells, in Acute Myeloid Leukemia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 08:47:00","doi":"10.21203/rs.3.rs-6523399/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-07-10T11:11:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-07-07T16:55:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-06-22T11:06:08+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-16T19:40:32+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-06-16T06:37:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-06-15T18:38:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-12T10:19:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-12T10:19:35+00:00","index":"","fulltext":""},{"type":"submitted","content":"Leukemia","date":"2025-06-11T19:24:56+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-04-25T10:25:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"leukemia","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"leu","sideBox":"Learn more about [Leukemia](http://www.nature.com/leu/)","snPcode":"41375","submissionUrl":"https://mts-leu.nature.com/cgi-bin/main.plex","title":"Leukemia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a987d942-0cb3-4e1b-ad05-b8922b3f99cc","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50075393,"name":"Health sciences/Health care/Therapeutics/Immunotherapy"},{"id":50075394,"name":"Biological sciences/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia"},{"id":50075395,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":50075396,"name":"Biological sciences/Stem cells/Haematopoietic stem cells"},{"id":50075397,"name":"Health sciences/Medical research/Preclinical research"}],"tags":[],"updatedAt":"2025-10-28T17:31:34+00:00","versionOfRecord":{"articleIdentity":"rs-6523399","link":"https://doi.org/10.1038/s41375-025-02764-7","journal":{"identity":"leukemia","isVorOnly":false,"title":"Leukemia"},"publishedOn":"2025-10-27 04:00:00","publishedOnDateReadable":"October 27th, 2025"},"versionCreatedAt":"2025-06-18 08:47:00","video":"","vorDoi":"10.1038/s41375-025-02764-7","vorDoiUrl":"https://doi.org/10.1038/s41375-025-02764-7","workflowStages":[]},"version":"v1","identity":"rs-6523399","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6523399","identity":"rs-6523399","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}