SLC27A2 is a potential immune biomarker for hematological tumors and significantly regulates the cell cycle progression of diffuse large B-cell lymphoma.

BACKGROUND: Research on the fatty acid metabolism related gene SLC27A2 is currently mainly focused on solid tumors, and its mechanism of action in hematological tumors has not been reported. METHOD: This study aims to explore the pathological and immune mechanisms of the fatty acid metabolism related gene SLC27A2 in hematological tumors and verify its functional role in hematological tumors through cell experiments to improve treatment decisions and clinical outcomes of hematological tumors. RESULT: This study identified the fatty acid metabolism related gene SLC27A2 as a common differentially expressed gene between DLBCL and AML. Immune microenvironment analysis showed that SLC27A2 was significantly positively correlated with T cell CD4 + , T cell CD8 + , endothelial cells, macrophages, and NK cells in DLBCL. In AML, there is a significant negative correlation between SLC27A2 and B cells, T cell CD8 + , and macrophages. SLC27A2 participates in the immune process of hematological tumors through T cell CD8 + and macrophages. The GESA results indicate that high expression of SLC27A2 is mainly involved in the fatty acid pathway, immune pathway, and cell cycle pathway of DLBCL. The low expression of SLC27A2 is mainly involved in the immune pathway of AML. Therefore, SLC27A2 is mainly involved in the pathological mechanisms of hematological tumors through immune pathways, and cell experiments have also confirmed that SLC27A2 is involved in the regulation of DLBCL cells. CONCLUSION: In summary, our research results comprehensively report for the first time the mechanism of action of SLC27A2 in the immune microenvironment of DLBCL and AML, and for the first time verify the cycle and apoptotic effects of the fatty acid related gene SLC27A2 in DLBCL cells through cell experiments. Research can help improve the treatment of AML and DLBCL patients.

Targeting IDH2R140Q and other neoantigens in acute myeloid leukemia.

ABSTRACT: For patients with high-risk or relapsed/refractory acute myeloid leukemia (AML), allogeneic stem cell transplantation (allo-HSCT) and the graft-versus-leukemia effect mediated by donor T cells, offer the best chance of long-term remission. However, the concurrent transfer of alloreactive T cells can lead to graft-versus-host disease that is associated with transplant-related morbidity and mortality. Furthermore, ∼60% of patients will ultimately relapse after allo-HSCT, thus, underscoring the need for novel therapeutic strategies that are safe and effective. In this study, we explored the feasibility of immunotherapeutically targeting neoantigens, which arise from recurrent nonsynonymous mutations in AML and thus represent attractive targets because they are exclusively present on the tumor. Focusing on 14 recurrent driver mutations across 8 genes found in AML, we investigated their immunogenicity in 23 individuals with diverse HLA profiles. We demonstrate the immunogenicity of AML neoantigens, with 17 of 23 (74%) reactive donors screened mounting a response. The most immunodominant neoantigens were IDH2R140Q (n = 11 of 17 responders), IDH1R132H (n = 7 of 17), and FLT3D835Y (n = 6 of 17). In-depth studies of IDH2R140Q-specific T cells revealed the presence of reactive CD4+ and CD8+ T cells capable of recognizing distinct mutant-specific epitopes restricted to different HLA alleles. These neo-T cells could selectively recognize and kill HLA-matched AML targets endogenously expressing IDH2R140Q both in vitro and in vivo. Overall, our findings support the clinical translation of neoantigen-specific T cells to treat relapsed/refractory AML.

Determination of Cuproptosis-related Subtypes, Development of a Prognostic Model, and Characterization of Tumor Microenvironment Infiltration in Acute Myeloid Leukemia.

BACKGROUND/AIM: Acute myeloid leukemia (AML) is a severe malignancy of the bone marrow marked by an abnormal accumulation of bone marrow precursors. Cuproptosis is a recently identified type of copper-dependent regulatory cell apoptosis that relies on mitochondrial respiration. However, its participation in the development of AML remains unclear. This study analyzed the association between cuproptosis-related genes and the prognosis of AML patients. MATERIALS AND METHODS: Cases of AML were acquired from TCGA, GEO, and TARGET and the molecular subgroups characterized by genes associated with cuproptosis, besides the associated cell infiltration of the tumor microenvironment (TME) were investigated. The cuproptosis score was developed using the minor absolute shrinkage and selection operator (LASSO) tool to evaluate the cuproptosis features of a single tumor sample. RESULTS: Two distinct molecular subgroups related to cuproptosis were discovered in AML with different prognoses. The cellular infiltration assay of TME showed immunological heterogeneity between the two subtypes. The cuproptosis score predicted tumor subgroups, immunity, and prognosis. A small cuproptosis value was marked by a good prognosis, whereas the anti-PD-1/PD-L1 immunotherapy group suggested the same cuproptosis group was related to an elevated immunotherapy potency. CONCLUSION: The cuproptosis score is a biomarker important for determining the molecular subgroups, prognosis, TME cell infiltration features, and immunotherapeutic efficacy of individuals with leukemia.

Epitope editing enables targeted immunotherapy of acute myeloid leukaemia.

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia1,2, the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens3-5. Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34+ HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning.

Immunotherapy in leukaemia.

Leukaemia is the common name for a group of malignant diseases of the haematopoietic system with complex classifications and characteristics. Remarkable progress has been made in basic research and preclinical studies for acute leukaemia compared to that of the many other types/subtypes of leukaemia, especially the exploration of the biological basis and application of immunotherapy in acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (B-ALL). In this review, we summarize the basic approaches to immunotherapy for leukaemia and focus on the research progress made in immunotherapy development for AML and ALL. Importantly, despite the advances made to date, big challenges still exist in the effectiveness of leukaemia immunotherapy, especially in AML. Therefore, we use AML as an example and summarize the mechanisms of tumour cell immune evasion, describe recently reported data and known therapeutic targets, and discuss the obstacles in finding suitable treatment targets and the results obtained in recent clinical trials for several types of single and combination immunotherapies, such as bispecific antibodies, cell therapies (CAR-T-cell treatment), and checkpoint blockade. Finally, we summarize novel immunotherapy strategies for treating lymphocytic leukaemia and clinical trial results.

Exogenous Antigen Upregulation Empowers Antibody Targeted Nanochemotherapy of Leukemia.

Acute myeloid leukemia (AML) is afflicted by a high-mortality rate and few treatment options. The lack of specific surface antigens severely hampers the development of targeted therapeutics and cell therapy. Here, it is shown that exogenous all-trans retinoic acid (ATRA) mediates selective and transient CD38 upregulation on leukemia cells by up to 20-fold, which enables high-efficiency targeted nanochemotherapy of leukemia with daratumumab antibody-directed polymersomal vincristine sulfate (DPV). Strikingly, treatment of two CD38-low expressing AML orthotopic models with ATRA and DPV portfolio strategies effectively eliminates circulating leukemia cells and leukemia invasion into bone marrow and organs, leading to exceptional survival benefits with 20-40% of mice becoming leukemia-free. The combination of exogenous CD38 upregulation and antibody-directed nanotherapeutics provides a unique and powerful targeted therapy for leukemia.

Activating STING1-dependent immune signaling in TP53 mutant and wild-type acute myeloid leukemia.

DNA methyltransferase inhibitors (DNMTis) reexpress hypermethylated genes in cancers and leukemias and also activate endogenous retroviruses (ERVs), leading to interferon (IFN) signaling, in a process known as viral mimicry. In the present study we show that in the subset of acute myeloid leukemias (AMLs) with mutations in TP53, associated with poor prognosis, DNMTis, important drugs for treatment of AML, enable expression of ERVs and IFN and inflammasome signaling in a STING-dependent manner. We previously reported that in solid tumors poly ADP ribose polymerase inhibitors (PARPis) combined with DNMTis to induce an IFN/inflammasome response that is dependent on STING1 and is mechanistically linked to generation of a homologous recombination defect (HRD). We now show that STING1 activity is actually increased in TP53 mutant compared with wild-type (WT) TP53 AML. Moreover, in TP53 mutant AML, STING1-dependent IFN/inflammatory signaling is increased by DNMTi treatment, whereas in AMLs with WT TP53, DNMTis alone have no effect. While combining DNMTis with PARPis increases IFN/inflammatory gene expression in WT TP53 AML cells, signaling induced in TP53 mutant AML is still several-fold higher. Notably, induction of HRD in both TP53 mutant and WT AMLs follows the pattern of STING1-dependent IFN and inflammatory signaling that we have observed with drug treatments. These findings increase our understanding of the mechanisms that underlie DNMTi + PARPi treatment, and also DNMTi combinations with immune therapies, suggesting a personalized approach that statifies by TP53 status, for use of such therapies, including potential immune activation of STING1 in AML and other cancers.

WT1-specific TCRs directed against newly identified peptides install antitumor reactivity against acute myeloid leukemia and ovarian carcinoma.

BACKGROUND: Transcription factor Wilms' tumor gene 1 (WT1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in cancer progression. In clinical trials, WT1 is targeted using peptide-based or dendritic cell-based vaccines and T-cell receptor (TCR)-based therapies. Antitumor reactivities were reported, but T-cell reactivity is hampered by self-tolerance to WT1 and limited number of WT1 peptides, which were thus far selected based on HLA peptide binding algorithms. METHODS: In this study, we have overcome both limitations by searching in the allogeneic T-cell repertoire of healthy donors for high-avidity WT1-specific T cells, specific for WT1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma samples. RESULTS: Using broad panels of malignant cells and healthy cell subsets, T-cell clones were selected that demonstrated potent and specific anti-WT1 T-cell reactivity against five of the eight newly identified WT1 peptides. Notably, T-cell clones for WT1 peptides previously used in clinical trials lacked reactivity against tumor cells, suggesting limited processing and presentation of these peptides. The TCR sequences of four T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against WT1-expressing solid tumor cell lines, primary acute myeloid leukemia (AML) blasts, and ovarian carcinoma patient samples. CONCLUSIONS: Our approach resulted in a set of naturally expressed WT1 peptides and four TCRs that are promising candidates for TCR gene transfer strategies in patients with WT1-expressing tumors, including AML and ovarian carcinoma.

CD70 CAR T cells in AML: Form follows function.

Using a multimodal approach toward developing a new CD70-targeted Chimeric antigen receptor (CAR) T cell in acute myeloid leukemia, Leick et al.1 report on their synergetic strategy, which incorporates both CAR T cell construct modifications with enhancement of leukemia antigen expression to improve CAR T cell functionality.

HCK is a Potential Prognostic Biomarker that Correlates with Immune Cell Infiltration in Acute Myeloid Leukemia.

Background: The tumor microenvironment (TME) plays a significant role in the progression and prognosis of acute myeloid leukemia (AML). This study is aimed at exploring TME-associated biomarkers and identify their potential mechanism in the microenvironment of AML. Method: In this study, the stromal, immune, and ESTIMATE scores of AML patients were evaluated with the ESTIMATE and CIBERSORT algorithms; then, the AML samples were divided into high- and low-score groups. We evaluated the association between clinicopathological characteristics, survival rate, and the stromal/immune/ESTIMATE scores. Furthermore, we identified TME-associated differentially expressed genes (DEGs) then carried out pathway enrichment analysis, protein-protein interaction (PPI) network, Cox regression analysis, and Kaplan-Meier survival analysis to select the most crucial genes. In addition, we further explored the potential mechanism of HCK in the AML microenvironment. Results: We identified 624 TME-associated DEGs and found that HCK was the most promising biomarker associated with AML. The results of the gene set enrichment analysis (GSEA) indicated that HCK was mainly involved in immune and inflammation-related signaling pathways. In addition, CIBERSORT analysis showed that HCK was closely related to tumor immune infiltration, with HCK expression associated with various infiltrating immune cells, including B cells, T cells, tumor-associated macrophages (TAM), NK cells, plasma cells, eosinophils, and neutrophils. Furthermore, HCK expression was closely related with ELN risk stratification in patients with AML. Conclusion: HCK could regulate immune cell infiltration in the microenvironment of AML and may act as a potential biomarker for the treatment and prognosis of AML patients.

Integrated Multiomic Profiling Identifies the Epigenetic Regulator PRC2 as a Therapeutic Target to Counteract Leukemia Immune Escape and Relapse.

Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse. SIGNIFICANCE: Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397.

Targeting an alternate Wilms' tumor antigen 1 peptide bypasses immunoproteasome dependency.

Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)-restricted T cell receptor (TCR) specific for a Wilms' tumor antigen 1 epitope, WT1126-134 (TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient's AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (ß1i), which is required for presentation of WT1126-134. An analysis of a second patient treated with TTCR-C4 demonstrated specific loss of AML cells coexpressing ß1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2-restricted WT137-45 epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37-45) killed the first patients' relapsed AML resistant to WT1126-134 targeting, as well as other primary AML, in vitro. TTCR37-45 controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

CAR T cells redirected to cell surface GRP78 display robust anti-acute myeloid leukemia activity and do not target hematopoietic progenitor cells.

Developing CAR T cells for acute myeloid leukemia (AML) has been hampered by a paucity of targets that are expressed on AML blasts and not on hematopoietic progenitor cells (HPCs). Here we demonstrate that GRP78 is expressed on the cell surface of primary AML blasts but not HPCs. To target GRP78, we generate T cell expressing a GRP78-specific peptide-based CAR, which show evidence of minimal fratricide post activation/transduction and antigen-dependent T cell differentiation. GRP78-CAR T cells recognize and kill GRP78-positive AML cells without toxicity to HPCs. In vivo, GRP78-CAR T cells have significant anti-AML activity. To prevent antigen-dependent T cell differentiation, we block CAR signaling and GRP78 cell surface expression post activation by using dasatinib during GRP78-CAR T cell manufacturing. This significantly improves their effector function in vitro and in vivo. Thus, targeting cell surface GRP78-positive AML with CAR T cells is feasible, and warrants further active exploration.

m6A-related lncRNAs predict prognosis and indicate immune microenvironment in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a complex hematologic malignancy. Survival rate of AML patients is low. N6-methyladenosine (m6A) and long non-coding RNAs (lncRNAs) play important roles in AML tumorigenesis and progression. However, the relationship between lncRNAs and biological characteristics of AML, as well as how lncRNAs influence the prognosis of AML patients, remain unclear. In this study. In this study, Pearson correlation analysis was used to identify lncRNAs related to m6A regulatory genes, namely m6A-related lncRNAs. And we analyzed their roles and prognostic values in AML. m6A-related lncRNAs associated with patient prognosis were screened using univariate Cox regression analysis, followed by systematic analysis of the relationship between these genes and AML clinicopathologic and biologic characteristics. Furthermore, we examined the characteristics of tumor immune microenvironment (TIME) using different IncRNA clustering models. Using LASSO regression, we identified the risk signals related to prognosis of AML patients. We then constructed and verified a risk model based on m6A-related lncRNAs for independent prediction of overall survival in AML patients. Our results indicate that risk scores, calculated based on risk-related signaling, were related to the clinicopathologic characteristics of AML and level of immune infiltration. Finally, we examined the expression level of TRAF3IP2-AS1 in patient samples through real-time polymerase chain reaction analysis and in GEO datasets, and we identified a interaction relationship between SRSF10 and TRAF3IP2-AS1 through in vitro assays. Our study shows that m6A-related lncRNAs, evaluated using the risk prediction model, can potentially be used to predict prognosis and design immunotherapy in AML patients.

Venetoclax enhances NK cell killing sensitivity of AML cells through the NKG2D/NKG2DL activation pathway.

BACKGROUND: Venetoclax, a selective B-cell lymphoma-2 (BCL2) inhibitor, has a potential therapeutic effect when combined with demethylating agents in the first-line setting of unfit elderly patients with acute myeloid leukaemia (AML); however, efficacy is still limited in refractory/recurrent AML. Therefore, exploration of a suitable novel treatment scheme is urgently needed.However, combining venetoclax with NK cell-based immunotherapy has not been studied. METHODS: The cytotoxicity of NK cell combined with venetoclax was assessed in vitro using flow cytometry. Venetoclax-induced natural killer group 2 member D (NKG2D) ligand (NKG2DL) expression was detected by flow cytometry and western blotting. Mechanisms underlying venetoclax-induced NKG2DL expression were found by GSE127200 analysis and investigated using real-time PCR (Q-PCR) and western blotting. RESULTS: Flow cytometric analysis showed that combining venetoclax with NK cells produced synergistic anti-leukaemia effects similar to those of venetoclax + azacitidine. Venetoclax could render AML cell lines and primary AML cells sensitive to NK cell killing by promoting NK cell degranulation, NK-AML cell recognition and NK cell secretion of interferon (IFN)-γ and granzyme B. The synergistic effect resulted from venetoclax-induced NKG2DL upregulation in AML cells and could be undermined by blocking NKG2D on NK cells. This finding suggests that venetoclax enhances NK cell killing activity by activating the NKG2D/NKG2DL ligand-receptor pathway. Furthermore, the nuclear factor-kappa-B (NFKB) signalling pathway was involved in venetoclax-induced NKG2DL upregulation. CONCLUSIONS: Collectively, our data confirm that venetoclax combined with NK cells induces synergistic AML cell cytolysis and preliminarily revealed that venetoclax could selectively induce NKG2DLs on AML cells via NFKB signalling pathway.

Asxl1 loss cooperates with oncogenic Nras in mice to reprogram the immune microenvironment and drive leukemic transformation.

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

Immune composition and its association with hematologic recovery after chemotherapeutic injury in acute myeloid leukemia.

Chemotherapy-induced bone marrow (BM) injury is a significant cause of morbidity and mortality in acute myeloid leukemia (AML). Time to hematologic recovery after standard ("7 + 3") myeloablative chemotherapy can vary considerably among patients, but the factors that drive or predict BM recovery remain incompletely understood. Here, we assessed the composition of innate and adaptive immune subsets in the regenerating BM (day 17) after induction chemotherapy and related it to hematologic recovery in AML. T cells, and in particular the CD4 central memory (CD4CM) T-cell subset, were significantly enriched in the BM after chemotherapy, suggesting the relative chemoresistance of cells providing long-term memory for systemic pathogens. In contrast, B cells and other hematopoietic subsets were depleted. Higher frequencies of the CD4CM T-cell subset were associated with delayed hematopoietic recovery, whereas a high frequency of natural killer (NK) cells was related to faster recovery of neutrophil counts. The NK/CD4CM ratio in the BM after chemotherapy was significantly associated with the time to subsequent neutrophil recovery (Spearman's ρ = -0.723, p < 0.001, false discovery rate <0.01). The data provide novel insights into adaptive immune cell recovery after injury and identify the NK/CD4CM index as a putative predictor of hematopoietic recovery in AML.

Systemic IL-15 promotes allogeneic cell rejection in patients treated with natural killer cell adoptive therapy.

Natural killer (NK) cells are a promising alternative to T cells for cancer immunotherapy. Adoptive therapies with allogeneic, cytokine-activated NK cells are being investigated in clinical trials. However, the optimal cytokine support after adoptive transfer to promote NK cell expansion, and persistence remains unclear. Correlative studies from 2 independent clinical trial cohorts treated with major histocompatibility complex-haploidentical NK cell therapy for relapsed/refractory acute myeloid leukemia revealed that cytokine support by systemic interleukin-15 (IL-15; N-803) resulted in reduced clinical activity, compared with IL-2. We hypothesized that the mechanism responsible was IL-15/N-803 promoting recipient CD8 T-cell activation that in turn accelerated donor NK cell rejection. This idea was supported by increased proliferating CD8+ T-cell numbers in patients treated with IL-15/N-803, compared with IL-2. Moreover, mixed lymphocyte reactions showed that IL-15/N-803 enhanced responder CD8 T-cell activation and proliferation, compared with IL-2 alone. Additionally, IL-15/N-803 accelerated the ability of responding T cells to kill stimulator-derived memory-like NK cells, demonstrating that additional IL-15 can hasten donor NK cell elimination. Thus, systemic IL-15 used to support allogeneic cell therapy may paradoxically limit their therapeutic window of opportunity and clinical activity. This study indicates that stimulating patient CD8 T-cell allo-rejection responses may critically limit allogeneic cellular therapy supported with IL-15. This trial was registered at www.clinicaltrials.gov as #NCT03050216 and #NCT01898793.