Immunomodulatory drugs (IMiDs) are anticancer drugs with immunomodulatory, anti-angiogenesis, anti-proliferative, and pro-apoptotic properties. IMiDs are currently used for the treatment of multiple myeloma, myelodysplastic syndrome, and B-cell lymphoma; however, little is known about efficacy in acute myeloid leukemia (AML). We proposed in this study to investigate the relevance of IMiDs therapy for AML treatment. We evaluated the effect of IMiDs on primary AML blasts (n = 24), and the impact in natural killer (NK) cell-mediated immunosurveillance of AML. Using primary AML cells and an immunodeficient mouse leukemia xenograft model, we showed that IMiDs induce AML cell death in vitro and impair leukemia progression in vivo. In addition, treatment of AML blasts with IMiDs resulted in enhanced allogeneic NK cell anti-leukemia reactivity. Treatment by pomalidomide of AML blasts enhanced lysis, degranulation, and cytokine production by primary allogeneic NK cells. Furthermore, the treatment with lenalidomide of patients with myeloid malignancies resulted in NK cell phenotypic changes similar to those observed in vitro. IMiDs increased CD56 and decreased NKp30, NKp46, and KIR2D expression on NK cells. Finally, AML blasts treatment with IMiDs induced phenotypic alterations including downregulation of HLA-class I. The effect of pomalidomide was not correlated with cereblon expression and A/G polymorphism in AML cells. Our data revealed, a yet unobserved, dual effects on AML affecting both AML survival and their sensitivity to NK immunotherapy using IMiDs. Our study encourages continuing investigation for the use of IMiDs in AML, especially in combination with conventional therapy or immunotherapy strategies.

Understanding immune alterations in cancer patients is a major challenge and requires precise phenotypic study of immune subsets. Improvement of knowledge regarding the biology of natural killer (NK) cells and technical advances leads to the generation of high dimensional dataset. High dimensional flow cytometry requires tools adapted to complex dataset analyses. This study presents an example of NK cell maturation analysis in Healthy Volunteers (HV) and patients with Acute Myeloid Leukemia (AML) with an automated procedure using the FLOCK algorithm. This procedure enabled to automatically identify NK cell subsets according to maturation profiles, with 2D mapping of a four-dimensional dataset. Differences were highlighted in AML patients compared to HV, with an overall increase of NK maturation. Among patients, a strong heterogeneity in NK cell maturation defined three distinct profiles. Overall, automatic gating with FLOCK algorithm is a recent procedure, which enables fast and reliable identification of cell populations from high-dimensional cytometry data. Such tools are necessary for immune subset characterization and standardization of data analyses. This tool is adapted to new immune cell subsets discovery, and may lead to a better knowledge of NK cell defects in cancer patients. Overall, 2D mapping of NK maturation profiles enabled fast and reliable identification of NK cell subsets.

The management of relapsed or refractory acute myeloid leukemia (AML) continues to be therapeutically challenging. Non-toxic immunotherapy approaches are needed to provide long-term anti-leukemic effects. The goal of this study was to determine whether activated T cells (ATCs) armed with bispecific antibodies (BiAbs) could target and lyse leukemic and leukemic stem cells (LSCs). Anti-CD3 × anti-CD123 BiAb (CD123Bi) and anti-CD3 × anti-CD33GO (gemtuzumab ozogamicin [GO]) BiAb (CD33GOBi) were used to arm ATCs to produce bispecific antibody armed activated T cells (designated CD123 BATs or CD33GO BATs) to target AML cell lines, peripheral blood mononuclear cells from AML patients, and in vivo treatment of AML in xenogeneic NSG mice engrafted with leukemic cells. BATs exhibited high levels of specific cytotoxicity directed at AML cell lines at low 1:1 or 1:2 effector-to-target (E:T) ratios and secrete Th1 cytokines upon target engagement. In vivo study in AML-engrafted NSG mice showed significantly prolonged survival in mice treated with CD33GO BATs (p < 0.0001) or CD123 BATs (p < 0.0089) compared to ATC-treated control mice. Patient samples containing leukemic blasts and LSCs when treated with CD33GO BATs or CD123 BATs for 18 h showed a significant reduction (50%-100%; p < 0.005) in blasts and 75%-100% reduction in LSCs (p < 0.005) in most cases compared to unarmed ATCs. This approach may provide a potent and non-toxic strategy to target AML blasts and LSCs and enhance chemo-responsiveness in older patients who are likely to develop recurrent diseases.

Chimeric antigen receptor (CAR)-T cell therapy has achieved unprecedented success in treating hematopoietic malignancies. However, this cell therapy is hampered in treating acute myeloid leukemia (AML) due to lack of ideal cell surface targets that only express on AML blasts and leukemia stem cells (LSCs) but not on normal hematopoietic stem cells (HSCs).

NLRP3 inflammasome has been reported to be associated with the pathogenesis of multiple solid tumors. However, the role of NLRP3 inflammasome in acute myeloid leukemia (AML) remains unclear. We showed that NLRP3 inflammasome is over-expressed and highly activated in AML bone marrow leukemia cells, which is correlated with poor prognosis. The activation of NLRP3 inflammasome in AML cells promotes leukemia cells proliferation, inhibits apoptosis and increases resistance to chemotherapy, while inactivation of NLRP3 by caspase-1 or NF-κB inhibitor shows leukemia-suppressing effects. Bayesian networks analysis and cell co-culture tests further suggest that NLRP3 inflammasome acts through IL-1β but not IL-18 in AML. Knocking down endogenous IL-1β or anti-IL-1β antibody inhibits leukemia cells whereas IL-1β cytokine enhances leukemia proliferation. In AML murine model, up-regulation of NLRP3 increases the leukemia burden in bone marrow, spleen and liver, and shortens the survival time; furthermore, knocking out NLRP3 inhibits leukemia progression. Collectively, all these evidences demonstrate that NLRP3 inflammasome promotes AML progression in an IL-1β dependent manner, and targeting NLRP3 inflammasome may provide a novel therapeutic option for AML.

DNA (cytosine-5)-methyltransferase 3A (DNMT3A)-mutated acute myeloid leukemia (AML) has a poor prognosis, but the exact mechanism is still unclear. Here, we aimed to explore the mechanism of immune escape in AML with DNMT3A mutation. We constructed a DNMT3A knockout clone and DNMT3A-R882H-mutated clones. RNA-seq results showed that transcription factors and macrophage inflammatory proteins were significantly downregulated in the DNMT3A mutant clones. KEGG enrichment and gene set enrichment analysis (GSEA) showed that a large number of genes were enriched in inflammatory immune-related pathways, such as the toll-like receptor signaling pathway. Therefore, we co-cultured AML cells with macrophages. The DNMT3A-mutated AML cells attenuated M1 macrophage polarization and resisted its killing effect in vitro and in vivo. In xenografts, the tumor volumes in the experimental group were significantly larger than those in the control group, and the proportion of M2 macrophages was significantly higher. After the co-culture, the increase in pro-inflammatory cytokine expression in the mutant cells was significantly lower than that in the control group, while that in immunosuppressive factors was not significantly different. In co-cultivated supernatants, the concentration of inflammatory factors in the experimental group was significantly lower than that in the control group, while that of immunosuppressive factors was significantly higher. Resistin significantly promoted the expression of inflammatory proteins in AML cells. It relieved the inhibitory effect of DNMT3A mutation, promoted the phenotypic recovery of the co-cultured macrophages, eliminated resistance, and regulated the immune microenvironment. Thus, resistin may serve as an ancillary drug for patients with DNMT3A-mutated AML.

CD44v6 chimeric antigen receptor T (CD44v6 CAR-T) cells demonstrate strong anti-tumor ability and safety in acute myeloid leukemia (AML). However, the expression of CD44v6 on T cells leads to transient fratricide and exhaustion of CD44v6 CAR-T cells, which affect the application of CD44v6 CAR-T. The exhaustion and function of T cells and CD44v6 expression of AML cells are associated with DNA methylation. Hypomethylating agents (HAMs) decitabine (Dec) and azacitidine (Aza) have been widely used to treat AML. Therefore, there may be synergy between CD44v6 CAR-T cells and HAMs in the treatment of AML.

CAR T cell approaches to effectively target AML and T-ALL without off-tumor effects on healthy myeloid or T cell compartments respectively are an unmet medical need. NKG2D-ligands are a promising target given their absence on healthy cells and surface expression in a wide range of malignancies. NKG2D-ligand expression has been reported in a substantial group of patients with AML along with evidence for prognostic significance. However, reports regarding the prevalence and density of NKG2D-ligand expression in AML vary and detailed studies to define whether low level expression is sufficient to trigger NKG2D-ligand directed CART cell responses are lacking. NKG2D ligand expression in T-ALL has not previously been interrogated. Here we report that NKG2D-ligands are expressed in T-ALL cell lines and primary T-ALL. We confirm that NKG2D-ligands are frequently surface expressed in primary AML, albeit at relatively low levels. Utilizing CAR T cells incorporating the natural immune receptor NKG2D as the antigen binding domain, we demonstrate striking in vitro activity of CAR T cells targeting NKG2D-ligands against AML and T-ALL cell lines and show that even low-level ligand expression in primary AML targets results in robust NKG2D-CAR activity. We found that NKG2D-ligand expression can be selectively enhanced in low-expressing AML cell lines and primary AML blasts via pharmacologic HDAC inhibition. Such pharmacologic NKG2D-ligand induction results in enhanced NKG2D-CAR anti-leukemic activity without affecting healthy PBMC, thereby providing rationale for the combination of HDAC-inhibitors with NKG2D-CAR T cell therapy as a potential strategy to achieve clinical NKG2D-CAR T cell efficacy in AML.

Notch receptors deeply influence T-cell development and differentiation, and their dysregulation represents a frequent causative event in "T-cell acute lymphoblastic leukemia" (T-ALL). "Myeloid-derived suppressor cells" (MDSCs) inhibit host immune responses in the tumor environment, favoring cancer progression, as reported in solid and hematologic tumors, with the notable exception of T-ALL. Here, we prove that Notch-signaling deregulation in immature T cells promotes CD11b+Gr-1+ MDSCs in the Notch3-transgenic murine model of T-ALL. Indeed, aberrant T cells from these mice can induce MDSCs in vitro, as well as in immunodeficient hosts. Conversely, anti-Gr1-mediated depletion of MDSCs in T-ALL-bearing mice reduces proliferation and expansion of malignant T cells. Interestingly, the coculture with Notch-dependent T-ALL cell lines, sustains the induction of human CD14+HLA-DRlow/neg MDSCs from healthy-donor PBMCs that are impaired upon exposure to gamma-secretase inhibitors. Notch-independent T-ALL cells do not induce MDSCs, suggesting that Notch-signaling activation is crucial for this process. Finally, in both murine and human models, IL-6 mediates MDSC induction, which is significantly reversed by treatment with neutralizing antibodies. Overall, our results unveil a novel role of Notch-deregulated T cells in modifying the T-ALL environment and represent a strong premise for the clinical assessment of MDSCs in T-ALL patients.

Chimeric antigen receptor (CAR) T therapies are being developed for acute myeloid leukemia (AML) on the basis of the results obtained for other haematological malignancies and the need of new treatments for relapsed and refractory AML. The biggest challenge of CART therapy for AML is to identify a specific target antigen, since antigens expressed in AML cells are usually shared with healthy haematopoietic stem cells (HSC). The concomitant expression of the target antigen on both tumour and HSC may lead to on-target/off-tumour toxicity. In this review, we guide researchers to design, develop, and translate to the clinic CART therapies for the treatment of AML. Specifically, we describe what issues have to be considered to design these therapies; what in vitro and in vivo assays can be used to prove their efficacy and safety; and what expertise and facilities are needed to treat and manage patients at the hospital.

CD4+CD25+Foxp3+ regulatory T cells (Tregs) accumulate in bone marrow microenvironment in acute myeloid leukemia (AML). However, little is known about how the tumor environment including tumor cells themselves affects this process. Here we demonstrated that AML cells expressed inducible T-cell costimulator ligand (ICOSL) that can provide costimulation through ICOS for the conversion and expansion of Tregs sustaining high Foxp3 and CD25 expression as well as a suppressive function. TNF-a stimulation up-regulated the expression of ICOSL. Furthermore, both the conversion and expansion of CD4+CD25+Foxp3+ T cells and CD4+ICOS+Foxp3+ T cells were induced by co-culture with AML cells overexpressed ICOSL. CD4+CD25+ICOS+ T cells possessed stronger ability to secrete IL-10 than CD4+CD25+ICOS- T cells. The mechanism by which IL-10 promoted the proliferation of AML cells was dependent on the activation of the Akt, Erk1/2, p38, and Stat3 signaling pathways. Blockade of ICOS signaling using anti-ICOSL antibody impaired the generation of Tregs and retarded the progression of an AML mice model injected with C1498 cells. The expression of ICOSL of patient AML cells and ICOS+ Tregs were found to be predictors for overall survival and disease-free survival in patients with AML, with ICOS+ Treg cell subset being a stronger predictor than total Tregs. These results suggest that ICOSL expression by AML cells may directly drive Treg expansion as a mechanism of immune evasion and ICOS+ Treg cell frequency is a better prognostic predictor in patients with AML.

Therapy with Tyrosine Kinase Inhibitors (TKI) aiming stable deep molecular response is the gold standard to treat Chronic Myeloid Leukemia (CML). NKT-like cells (CD3+CD56+) combine characteristics of T and NK cells. The physiopathological role of these cells remains unknown although the literature refers their association with inflammation, autoimmune diseases, and cancer. Since the information regarding the role of NKT-like cells in CML is rare, we aimed at the characterization of these cells in CML patients treated with TKIs. Peripheral blood NKT-like cells from 48 CML patients and 40 healthy donors were analyzed by multiparametric flow cytometry. Functional tests consisting of co-culture with leukemic target cells (K562 cell line) were used to measure degranulation and cytokine production. Our results revealed that NKT-like cells are decreased in treated CML patients, although they present increased expression of activation markers (CD69 and HLA-DR), increased degranulation (CD107a) and impaired IFN-γ production. Significantly alterations on the expression of tumor recognition (NCRs and NKp80), and immune regulation receptors (LAG-3, TIM-3, and CD137) by NKT-like cells were observed in CML patients. Second generation TKIs increased cell activation (CD69) and decreased expression of NKp44 and NKp80 by NKT-like cells from CML patients when compared to Imatinib. CML patients that achieved deep molecular response (MR4.5) presented downregulation of NKp44 and LAG-3. Further studies are needed to clarify the role of these cells as biomarkers of therapy response and also to evaluate their value for discrimination of better candidates for sustained treatment-free remission after TKI discontinuation.

Natural killer (NK) cells are key innate immunity effectors that play a major role in malignant cell destruction. Based on expression patterns of CD16, CD56, CD57, and CD94, three distinct NK cell maturation stages have been described, which differ in terms of cytokine secretion, tissue migration, and the ability to kill target cells. Our study addressed NK cell maturation in bone marrow under three conditions: a normal developmental environment, during pre-leukemic state (myelodysplastic syndrome, MDS), and during leukemic transformation (acute myeloblastic leukemia, AML). In this study, we used a new tool to perform multicolor flow cytometry data analysis, based on principal component analysis, which allowed the unsupervised, accurate discrimination of immature, mature, and hypermature NK subpopulations. An impaired NK/T cell distribution was observed in the MDS bone marrow microenvironment compared with the normal and AML settings, and a phenotypic shift from the mature to the immature state was observed in NK cells under both the MDS and AML conditions. Furthermore, an impaired NK cell antitumor response, resulting in changes in NK cell receptor expression (CD159a, CD158a, CD158b, and CD158e1), was observed under MDS and AML conditions compared with the normal condition. The results of this study provide evidence for the failure of this arm of the immune response during the pathogenesis of myeloid malignancies. NK cell subpopulations display a heterogeneous and discordant dynamic on the spectrum between normal and pathological conditions. MDS does not appear to be a simple, intermediate stage but rather serves as a decisive step for the mounting of an efficient or ineffective immune response, leading to either the removal of the tumor cells or to malignancy.

Intratumoral accumulation of CD4+CD25+Foxp3+ regulatory T (Treg) cells occurs in acute myeloid leukemia (AML), but little is known about the role of tumor cells themselves in this process. Here, we showed that an immune checkpoint PD-L1 expressed by AML cells promoted the conversion and expansion of Treg cells sustaining high expression of Foxp3 and PD-1 as well as a suppressive function. Furthermore, an AML cell line HEL overexpressed PD-L1 promoted the conversion and expansion of Treg cells and CD4+PD-1+Foxp3+ T (PD-1+Treg) cells from the conventional CD4+ T cells. CD4+CD25highPD-1+ T cells secreted more IL-10 production than CD4+CD25highPD-1- T cells. IL-35, another cytokine secreted by Treg cells, promoted the proliferation of HL-60 cells and enhanced chemoresistance to cytarabine. Blockade of PD-1 signaling using anti-PD-L1 antibody dramatically impaired the generation of Treg cells and sharply retarded the progression of a murine AML model injected with C1498 cells. The frequency of intratumoral PD-1+ Treg cells was capable of predicting patient survival in patients with AML. In conclusion, our data suggest that PD-L1 expression by AML cells may directly drive Treg cell expansion as a mechanism of immune evasion and the frequency of PD-1+ Treg cells is a potential prognostic predictor in patients with AML.

Accumulating evidence highlights natural killer (NK) cell parameters as potential prognostic factors in cancer patients, which provides a strong rationale for developing therapeutic strategies aiming at restoring NK cell. However, reaching this point warrants better characterization of tumor-induced NK cell alterations. Our group recently reported heterogeneous NK maturation in acute myeloid leukemia (AML) patients. However, the clinical significance of such observations remained to be assessed on a larger cohort of patients. NK maturation based on expression of CD56, CD57, and KIR was assessed by flow cytometry in newly diagnosed AML patients (N = 87 patients from GOELAMS-LAM-IR-2006 multicenter trial). Clinical outcome was evaluated with regard to NK maturation profiles. Unsupervised integrated analysis of NK maturation markers confirmed the existence of three distinct groups of patients [hypomaturation (24.1%), intermediate maturation (66.7%), and hypermaturation (9.2%)]. In univariate analysis, significant differences in overall survival (OS) (P = 0.0006) and relapse-free survival (RFS) (P < 0.0001) were observed among these different groups. Patients with hypomaturation profile had reduced OS, with 3-year OS rates of 12.5 vs 57.1 and 57.4% for patients with intermediate and hypermaturation, respectively. Consistently, patients with hypomaturation profile had reduced RFS, with 3-year RFS rates of 0 vs 52.6 and 73.3% for patients with intermediate and hypermaturation, respectively. In multivariate Cox regression models, NK hypomaturation remained significantly associated with reduced OS and RFS, independent of other factors [hazard ratio (HR) = 4.15, P = 0.004 and HR = 8.23, P = 0.003, respectively]. NK maturation defects were further explored by mass cytometry and revealed that NK hypomaturation profile is associated with a reduced frequency of memory-like NK cells. In conclusion, besides classical alterations of NK triggering and inhibitory receptors expression in AML, we confirm that the homeostasis of NK maturation can be modified in the context of AML, notably with a deep maturation blockade in almost 10% patients.

In acute myeloid leukemia (AML), malignant stem cells hijack the normal bone marrow niche where they are largely protected from the current therapeutic approaches. Thus, eradicating these progenitors is the ultimate challenge in the treatment of this disease. Specifically, the development of chimeric antigen receptors (CARs) against distinct mesenchymal stromal cell subpopulations involved in the maintenance of leukemic stem cells within the malignant bone marrow microenvironment could represent a new strategy to improve CAR T-cell therapy efficacy, which is still unsuccessful in AML. As a proof of concept, we generated a novel prototype of Tandem CAR, with one specificity directed against the leukemic cell marker CD33 and the other against the mesenchymal stromal cell marker CD146, demonstrating its capability of simultaneously targeting two different cell types in a 2D co-culture system. Interestingly, we could also observe an in vitro inhibition of CAR T cell functionality mediated by stromal cells, particularly in later effector functions, such as reduction of interferon-gamma and interleukin-2 release and impaired proliferation of the CAR+ effector Cytokine-Induced Killer (CIK) cells. Taken together, these data demonstrate the feasibility of a dual targeting model against two molecules, which are expressed on two different target cells, but also highlight the immunomodulatory effect on CAR CIK cells exerted by stromal cells, confirming that the niche could be an obstacle to the efficacy of CAR T cells. This aspect should be considered in the development of novel CAR T cell approaches directed against the AML bone marrow niche.

Chronic myeloid leukemia (CML) in childhood and adolescence is a rare malignancy that can successfully be treated with the tyrosine kinase inhibitor (TKI) imatinib. According to the current experience, treatment is necessary for years and, in the majority of cases, a lifelong approach is required to control the malignant disease. To what extent imatinib causes immunosuppression in different age cohorts is a controversial discussion. According to general medical recommendations, live vaccines are contraindicated in individuals treated with imatinib. However, a recent increase in the number of globally reported cases of measles has been observed and continues to rise. Due to the high contagiousness of the virus, near-perfect vaccination coverage (herd immunity of 93 to 95%) is required to effectively protect against measles resurgence-a scenario that is not realistic in many countries. When four teenagers with CML (median age 13 years, range 12-15) who were enrolled into pediatric trial CML-paed II while on imatinib treatment (median treatment duration 36 months, range 11-84) were identified without protective measles and/or varicella titers, we carefully balanced the risks of a live vaccination under immunosuppressive TKI medication against the benefit of being protected. The patients underwent live vaccination with the live attenuated vaccines M-M-RVAX Pro® and Varivax® simultaneously (Patient #1), Priorix® and Varilix® consecutively (Patient #2), and Priorix® (Patients #3 and #4). While the first three patients were vaccinated while receiving TKI therapy, treatment with imatinib was interrupted in patient #4 for 1 week prior and 2 weeks after vaccination. Patients #1 and #3 reacted with stable long-term seroconversion. In Patient #2, serum titer conversion against measles and varicella could not be demonstrated and thus revaccination with Priorix® and Varilix® was performed 3 years later. However, protective titers did not develop or were lost again. Patient #4 also lost protective titers against measles when assessed 10 months after vaccination, but revaccination resulted in stable seroprotective titers over 12 months after the last vaccination during ongoing imatinib treatment. We conclude that in all patients, the safety of live vaccines could be documented, as no acute or late adverse events were observed. However, in line with observations that memory B-cells are lost under exposure to imatinib, revaccination may become necessary (two out of four patients in this small series lost their seroprotection). Considering that the number of cases is very small, we also suggest some criteria for decision-making regarding live vaccinations of CML patients treated with imatinib.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous bone marrow malignancy, the only curative treatment being intensive chemotherapy eventually in combination with allogeneic stem cell transplantation. Both the AML and their neighboring stromal cells show constitutive chemokine release, but chemokines seem to function as regulators of AML cell proliferation only for a subset of patients. Chemokine targeting is therefore considered not only for immunosuppression in allotransplanted patients but also as a possible antileukemic strategy in combination with intensive chemotherapy or as part of disease-stabilizing treatment at least for the subset of patients with chemokine-responsive AML cells. In this study, we characterized more in detail the leukemia cell phenotype of the chemokine-responsive patients. We investigated primary AML cells derived from 79 unselected patients. Standardized in vitro suspension cultures were used to investigate AML cell proliferation, and global gene expression profiles were compared for chemokine responders and non-responders identified through the proliferation assays. CCL28-induced growth modulation was used as marker of chemokine responsiveness, and 38 patients were then classified as chemokine-responsive. The effects of exogenous CCL28 (growth inhibition/enhancement/no effect) thus differed among patients and was also dependent on the presence of exogenous hematopoietic growth factors as well as constitutive AML cell cytokine release. The effect of CCR1 inhibition in the presence of chemokine-secreting mesenchymal stem cells also differed among patients. Chemokine-responsive AML cells showed altered expression of genes important for (i) epigenetic transcriptional regulation, particularly lysine acetylation; (ii) helicase activity, especially DExD/H RNA helicases; and (iii) angioregulatory proteins important for integrin binding. Thus, chemokine responsiveness is part of a complex AML cell phenotype with regard to extracellular communication and transcriptional regulation. Chemokine targeting in chemokine-responsive patients may thereby alter AML cell trafficking and increase their susceptibility toward antileukemic treatment, e.g., conventional chemotherapy or targeting of other phenotypic characteristics of the chemokine-responsive cells.

Antibody-based immunotherapy is increasingly employed to treat acute lymphoblastic leukemia (ALL) patients. Many T-ALL cells express CD38 on their surface, which can be targeted by the CD38 antibody daratumumab (DARA), approved for the treatment of multiple myeloma. Tumor cell killing by myeloid cells is relevant for the efficacy of many therapeutic antibodies and can be more efficacious with human IgA than with IgG antibodies. This is demonstrated here by investigating antibody-dependent cellular phagocytosis (ADCP) by macrophages and antibody-dependent cell-mediated cytotoxicity (ADCC) by polymorphonuclear (PMN) cells using DARA (human IgG1) and an IgA2 isotype switch variant (DARA-IgA2) against T-ALL cell lines and primary patient-derived tumor cells. ADCP and ADCC are negatively regulated by interactions between CD47 on tumor cells and signal regulatory protein alpha (SIRPα) on effector cells. In order to investigate the impact of this myeloid checkpoint on T-ALL cell killing, CD47 and glutaminyl-peptide cyclotransferase like (QPCTL) knock-out T-ALL cells were employed. QPTCL is an enzymatic posttranslational modifier of CD47 activity, which can be targeted by small molecule inhibitors. Additionally, we used an IgG2σ variant of the CD47 blocking antibody magrolimab, which is in advanced clinical development. Moreover, treatment of T-ALL cells with all-trans retinoic acid (ATRA) increased CD38 expression leading to further enhanced ADCP and ADCC, particularly when DARA-IgA2 was applied. These studies demonstrate that myeloid checkpoint blockade in combination with IgA2 variants of CD38 antibodies deserves further evaluation for T-ALL immunotherapy.

Natural killer (NK) cells are a very important component of the innate immune response involved in the lysis of virus infected and tumor cells. Aging has a profound impact in the frequency, phenotype and function of NK cells. Chronic Myeloid Leukemia (CML) is caused by the BCR-ABL gene formation encoding aberrant oncoprotein tyrosine kinase. Treatment with tyrosine kinase inhibitors (TKIs) induces durable deep molecular response. The response to treatment and life expectancy is lower in older patients with chronic phase of CML than in younger patients. In this work we analyse NK cells from TKI-treated CML patients and healthy controls stratified according to age. We have analyzed the expression of NK receptors, activation markers, NK cell differentiation in CD56bright and CD56dim NK cell subsets and the expression of CD107a and IFN-γ in NK cells stimulated with K562. Whereas significant differences on the phenotype and function of NK cells were found between middle-aged (35-65 years old) and elderly (older than 65) healthy individuals, NK cells from TKI-treated CML patients do not show significant differences related with age in most parameters studied, indicating that age is not a limitation of the NK cell recovery after treatment with TKI. Our results also revealed differences in the expression of NK receptors, activation markers and functional assays in NK cells from TKI-treated CML patients compared with age-matched healthy controls. These results highlight the relevance of NK cells in TKI-treated patients and the need of an extensive analysis of the effect of aging on NK cell phenotype and function in these patients in order to define new NK-cell based strategies directed to control CML progression and achieve long-term disease remission after TKI cessation.