Natural killer (NK) cells are an emerging cellular immunotherapy for patients with acute myeloid leukemia (AML); however, the best approach to maximize NK cell antileukemia potential is unclear. Cytokine-induced memory-like NK cells differentiate after a brief preactivation with interleukin-12 (IL-12), IL-15, and IL-18 and exhibit enhanced responses to cytokine or activating receptor restimulation for weeks to months after preactivation. We hypothesized that memory-like NK cells exhibit enhanced antileukemia functionality. We demonstrated that human memory-like NK cells have enhanced interferon-γ production and cytotoxicity against leukemia cell lines or primary human AML blasts in vitro. Using mass cytometry, we found that memory-like NK cell functional responses were triggered against primary AML blasts, regardless of killer cell immunoglobulin-like receptor (KIR) to KIR-ligand interactions. In addition, multidimensional analyses identified distinct phenotypes of control and memory-like NK cells from the same individuals. Human memory-like NK cells xenografted into mice substantially reduced AML burden in vivo and improved overall survival. In the context of a first-in-human phase 1 clinical trial, adoptively transferred memory-like NK cells proliferated and expanded in AML patients and demonstrated robust responses against leukemia targets. Clinical responses were observed in five of nine evaluable patients, including four complete remissions. Thus, harnessing cytokine-induced memory-like NK cell responses represents a promising translational immunotherapy approach for patients with AML.

New therapies for patients with hematologic malignancies who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) are needed. Interleukin 15 (IL-15) is a cytokine that stimulates CD8 T-cell and natural killer (NK) cell antitumor responses, and we hypothesized this cytokine may augment antileukemia/antilymphoma immunity in vivo. To test this, we performed a first-in-human multicenter phase 1 trial of the IL-15 superagonist complex ALT-803 in patients who relapsed >60 days after allo-HCT. ALT-803 was administered to 33 patients via the IV or subcutaneous (SQ) routes once weekly for 4 doses (dose levels of 1, 3, 6, and 10 μg/kg). ALT-803 was well tolerated, and no dose-limiting toxicities or treatment-emergent graft-versus-host disease requiring systemic therapy was observed in this clinical setting. Adverse events following IV administration included constitutional symptoms temporally related to increased serum IL-6 and interferon-γ. To mitigate these effects, the SQ route was tested. SQ delivery resulted in self-limited injection site rashes infiltrated with lymphocytes without acute constitutional symptoms. Pharmacokinetic analysis revealed prolonged (>96 hour) serum concentrations following SQ, but not IV, injection. ALT-803 stimulated the activation, proliferation, and expansion of NK cells and CD8 T cells without increasing regulatory T cells. Responses were observed in 19% of evaluable patients, including 1 complete remission lasting 7 months. Thus, ALT-803 is a safe, well-tolerated agent that significantly increased NK and CD8 T cell numbers and function. This immunostimulatory IL-15 superagonist warrants further investigation to augment antitumor immunity alone and combined with other immunotherapies. This trial was registered at www.clinicaltrials.gov as #NCT01885897.

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• FLs harbor more recurrent mutations in the BCR signaling pathway, SWI/SNF complex, and histone genes than previously known.• Novel recurrent mutations affecting BTK, SYK, and HVCN1 may have therapeutic and prognostic implications for FL.Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma, yet it remains only partially characterized at the genomic level. To improve our understanding of the genetic underpinnings of this incurable and clinically heterogeneous disease, whole-exome sequencing was performed on tumor/normal pairs from a discovery cohort of 24 patients with FL. Using these data and mutations identified in other B-cell malignancies, 1716 genes were sequenced in 113 FL tumor samples from 105 primarily treatment-naive individuals. We identified 39 genes that were mutated significantly above background mutation rates. CREBBP mutations were associated with inferior PFS. In contrast, mutations in previously unreported HVCN1, a voltage-gated proton channel-encoding gene and B-cell receptor signaling modulator, were associated with improved PFS. In total, 47 (44.8%) patients harbor mutations in the interconnected B-cell receptor (BCR) and CXCR4 signaling pathways. Histone gene mutations were more frequent than previously reported (identified in 43.8% of patients) and often co-occurred (17.1% of patients). A novel, recurrent hotspot was identified at a posttranslationally modified residue in the histone H2B family. This study expands the number of mutated genes described in several known signaling pathways and complexes involved in lymphoma pathogenesis (BCR, Notch, SWitch/sucrose nonfermentable (SWI/SNF), vacuolar ATPases) and identified novel recurrent mutations (EGR1/2, POU2AF1, BTK, ZNF608, HVCN1) that require further investigation in the context of FL biology, prognosis, and treatment. (Blood. 2017;129(4):473-483)

NK cells are a promising cellular immunotherapy for cancer. Cytokine-induced memory-like (ML) NK cells differentiate after activation with IL-12, IL-15, and IL-18, exhibit potent anti-tumor responses, and safely induce complete remissions in patients with leukemia. However, many cancers are not fully recognized via NK cell receptors. Chimeric antigen receptors (CAR) have been utilized to enhance tumor-specific recognition by effector lymphocytes. We hypothesized that memory-like differentiation and CAR-engineering would result in complementary improvements in NK cell responses against NK-resistant cancers. To test this idea, peripheral blood ML NK cells were modified to express an anti-CD19 CAR (19-CAR-ML), which displayed significantly increased IFN-γ production, degranulation, and specific killing against NK-resistant lymphoma lines and primary targets, compared to non-specific control CAR-ML NK cells or conventional CAR NK cells. The 19-CAR and ML responses were synergistic, CAR-specific, and required ITAM signaling. Furthermore, 19-CAR-ML NK generated from lymphoma patients exhibited improved responses against their autologous lymphoma. 19-CAR-ML NK cells controlled lymphoma burden in vivo and improved survival in human xenograft models. Thus, CAR engineering of ML NK cells enhanced responses against resistant cancers and warrant further investigation, with the potential to broaden ML NK cell recognition against a variety of NK cell-resistant tumors.

Atopic dermatitis (AD) is a widespread, chronic skin disease associated with aberrant allergic inflammation. Current treatments involve either broad or targeted immunosuppression strategies. However, enhancing the immune system to control disease remains untested. We demonstrate that patients with AD harbor a blood natural killer (NK) cell deficiency that both has diagnostic value and improves with therapy. Multidimensional protein and RNA profiling revealed subset-level changes associated with enhanced NK cell death. Murine NK cell deficiency was associated with enhanced type 2 inflammation in the skin, suggesting that NK cells play a critical immunoregulatory role in this context. On the basis of these findings, we used an NK cell–boosting interleukin-15 (IL-15) superagonist and observed marked improvement in AD-like disease in mice. These findings reveal a previously unrecognized application of IL-15 superagonism, currently in development for cancer immunotherapy, as an immunotherapeutic strategy for AD.

Myeloid-derived cells play important modulatory and effector roles in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells, composed of monocytic (MO) and polymorphonuclear (PMN) fractions, which can suppress T cell activities in EAE. Their role in MS remains poorly characterized. We found decreased numbers of circulating MDSCs, driven by lower frequencies of the MO-MDSCs, and higher MDSC expression of microRNA miR-223 in MS versus healthy subjects. To gain mechanistic insights, we interrogated the EAE model. MiR-223 knock out (miR-223−/−) mice developed less severe EAE with increased MDSC numbers in the spleen and spinal cord compared to littermate controls. MiR-223−/− MO-MDSCs suppressed T cell proliferation and cytokine production in vitro and EAE in vivo more than wild-type MO-MDSCs. They also displayed an increased expression of critical mediators of MDSC suppressive function, Arginase-1(Arg1), and the signal transducer and activator of transcription 3 (Stat3), which herein, we demonstrate being an miR-223 target gene. Consistently, MDSCs from MS patients displayed decreased STAT3 and ARG1 expression compared with healthy controls, suggesting that circulating MDSCs in MS are not only reduced in numbers but also less suppressive. These results support a critical role for miR-223 in modulating MDSC biology in EAE and in MS and suggest potential novel therapeutic applications.

Tolerizing mechanisms within the host and tumor microenvironment inhibit T cell effector functions that can control cancer. These mechanisms blunt adoptive immunotherapy with infused T cells due to a complex array of signals that determine T cell tolerance, survival, or deletion. Ligation of the negative regulatory receptors CTLA4, PD-1(PDCD1) or LAG3 on T cells normally hinders their response to antigen through non-redundant biochemical processes that interfere with stimulatory pathways. In this study, we used an established mouse model of T cell tolerance to define the roles of these inhibitory receptors in regulating CD8+ T cell tolerance during adoptive immunotherapy to treat leukemia. Blocking CTLA4 and PD-1 in vivo combined to promote survival of transferred T cells despite powerful deletional signals that mediate Bim (BCL2L11)-dependent apoptosis. However, this dual blockade was not optimal for stimulating effector function by responding T cells, which required the additional blockade of LAG3 to induce full expansion and allow the acquisition of robust cytolytic activity. Thus, the cooperation of multiple distinct regulatory pathways was needed for the survival and effector differentiation of adoptively transferred tumor-reactive CD8+ T cells. Our work defines the immune escape pathways where simultaneous blockade could yield durable immunotherapeutic responses that can eradicate disseminated leukemia.