Catherine R. Keppel

Cytokine activation induces human memory-like NK cells

Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

A phase 2 multicenter study of lenalidomide in relapsed or refractory classical Hodgkin lymphoma

Relapsed or refractory (rel/ref) classical Hodgkin lymphoma (cHL) remains a clinical challenge, with limited effective treatment options available after stem cell transplantation. In a multicenter phase 2 study, the efficacy of lenalidomide in rel/ref cHL patients was evaluated at a dose of 25 mg/d on days 1-21 of a 28-day cycle. Patients remained on lenalidomide until disease progression or an unacceptable adverse event (AE) occurred. Thirty-eight cHL patients were enrolled with a median of 4 (range, 2-9) prior therapies; 87% had undergone prior stem cell transplantation and 55% of patients did not respond to their last prior therapy. Of 36 evaluable patients, responses were 1 complete remission (CR), 6 partial remissions (PRs), and 5 patients with stable disease (SD) for ≥ 6 months resulting in an International Working Committee (IWC) objective overall response rate (ORR) of 19% and a cytostatic ORR of 33%. Decreased chemokine (CCL17 and CCL22) plasma levels at 2 weeks were associated with a subsequent response. The treatment was well tolerated, and the most common grade 3/4 AEs were neutropenia (47%), anemia (29%), and thrombocytopenia (18%). Four patients discontinued lenalidomide because of rash, elevated transaminases/bilirubin, and cytopenias. We provide preliminary evidence of lenalidomides activity in patients with rel/ref cHL, and therefore exploration of lenalidomide in combination with other active agents is warranted. This trial is registered at www.ClinicalTrials.gov as NCT00540007.

MicroRNA-Deficient NK Cells Exhibit Decreased Survival but Enhanced Function

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3′UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3′UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.

The IL-15-Based ALT-803 Complex Enhances FcγRIIIa-Triggered NK Cell Responses and in Vivo Clearance of B Cell Lymphomas

Purpose: Anti-CD20 monoclonal antibodies (mAb) are an important immunotherapy for B-cell lymphoma, and provide evidence that the immune system may be harnessed as an effective lymphoma treatment approach. ALT-803 is a superagonist IL-15 mutant and IL-15Rα–Fc fusion complex that activates the IL-15 receptor constitutively expressed on natural killer (NK) cells. We hypothesized that ALT-803 would enhance anti–CD20 mAb-directed NK-cell responses and antibody-dependent cellular cytotoxicity (ADCC). Experimental Design: We tested this hypothesis by adding ALT-803 immunostimulation to anti-CD20 mAb triggering of NK cells in vitro and in vivo. Cell lines and primary human lymphoma cells were utilized as targets for primary human NK cells. Two complementary in vivo mouse models were used, which included human NK-cell xenografts in NOD/SCID-γc−/− mice. Results: We demonstrate that short-term ALT-803 stimulation significantly increased degranulation, IFNγ production, and ADCC by human NK cells against B-cell lymphoma cell lines or primary follicular lymphoma cells. ALT-803 augmented cytotoxicity and the expression of granzyme B and perforin, providing one potential mechanism for this enhanced functionality. Moreover, in two distinct in vivo B-cell lymphoma models, the addition of ALT-803 to anti-CD20 mAb therapy resulted in significantly reduced tumor cell burden and increased survival. Long-term ALT-803 stimulation of human NK cells induced proliferation and NK-cell subset changes with preserved ADCC. Conclusions: ALT-803 represents a novel immunostimulatory drug that enhances NK-cell antilymphoma responses in vitro and in vivo, thereby supporting the clinical investigation of ALT-803 plus anti-CD20 mAbs in patients with indolent B-cell lymphoma. Clin Cancer Res; 22(3); 596–608. ©2015 AACR.

Additional evidence of a locus for complex febrile and afebrile seizures on chromosome 12q22-23.3

Sir: Febrile seizures are common, affecting 2–5% of all children worldwide [1]. Multiple loci for autosomal dominant febrile seizures have been reported (8q, 19p, 2q, 6q, and 5q), in addition to a locus on chromosome 12q22 identified in a large Belgian family with familial temporal lobe epilepsy and febrile seizures [2]. We identified a North American Caucasian family with seven affected individuals with febrile seizures (Fig. 1). Four individuals had febrile status epilepticus; three required intensive care unit hospitalization. Three of these individuals also had afebrile seizures during childhood. Seizures were generalized tonic or tonic–clonic, although two individuals had febrile seizures with focal features. Two affected individuals were born prematurely. The first (IV:10), born at 28 weeks, has spastic quadraplegia and frequent febrile and afebrile seizures, which ceased at age 6 years (now seizure-free at age 34). The second (V:1), born at 34 weeks, had normal development until age 2 following status epilepticus. He now has significant global developmental delay and parietal T2 signal abnormalities consistent with hypoxic or seizure-induced injury on brain magnetic resonance images (MRI; normal prior to status epilepticus). Electroencephalogram and brain MRI were performed in one other individual and were normal. Segregation of markers flanking seven known febrile seizure loci (GEFS+1-3 and FEB1-5) with disease was tested. Logarithm of odds (LOD) scores were <−2 at each locus, providing evidence for the exclusion of linkage. At the time of study, the chromosome 12q locus was not yet published, and a genome-wide scan at 18 cM resolution was performed. Linkage analysis was performed with MLINK assuming autosomal dominant inheritance, 80% penetrance, disease gene frequency of 0.1%, and phenocopy rate of 3%. Two loci yielded two-point LOD scores >2 (D12S1300 [LOD=2.40 at theta=0] and PAH [LOD=2.27 at theta=0]). Two other loci yielded two-point LOD scores >1 (D11S2371 [LOD=1.45 at theta=0] and D7S559 [LOD=1.23 at theta= 0]); additional nearby markers did not yield greater LOD scores. Additional chromosome 12 markers were genotyped, including D12S338, which yielded LOD=2.55 at theta=0. Haplotype reconstruction indicated that affected individuals and two obligate carriers shared a common chromosome 12q haplotype (Fig. 1). Obligate recombinants in individual IV:4 defined the candidate region centromerically at D12S297 (65 cM) and telomerically at D12S2070 (125 cM) in individual V:4. This mapped the disease gene Neurogenetics (2007) 8:61–63 DOI 10.1007/s10048-006-0063-z