Abstract: Autosomal recessive mutations in UNC13D, the gene that encodes Munc13-4, are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Munc13-4 expression is obligatory for exocytosis of lytic granules, facilitating cytotoxicity by T cells and natural killer (NK) cells. The mechanisms regulating Munc13-4 expression are unknown. Here, we report that Munc13-4 is highly expressed in differentiated human NK cells and effector CD8+ T lymphocytes. A UNC13D c.118-308C & gt;T mutation, causative of FHL3, disrupted binding of the ETS family member ELF1 to a conserved intronic sequence. This mutation impairs UNC13D intron 1 recruitment of STAT4 and the chromatin remodeling complex component BRG1, diminishing active histone modifications at the locus. The intronic sequence acted as an overall enhancer of Munc13-4 expression in cytotoxic lymphocytes in addition to representing an alternative promoter encoding a novel Munc13-4 isoform. Mechanistically, T cell receptor engagement facilitated STAT4-dependent Munc13-4 expression in naive CD8+ T lymphocytes. Collectively, our data demonstrates how chromatin remodeling within an evolutionarily conserved regulatory element in intron 1 of UNC13D regulates the induction of Munc13-4 expression in cytotoxic lymphocytes and suggests that an alternative Munc13-4 isoform is required for lymphocyte cytotoxicity. Thus, mutations associated with primary immunodeficiencies may cause disease by disrupting transcription factor binding.

Abstract: With an array of activating and inhibitory receptors, natural killer (NK) cells can specifically eradicate infected and transformed cells. Target cell killing is achieved through directed release of lytic granules. Recognition of target cells also induces production of chemokines and cytokines that can coordinate immune responses. Upon contact with susceptible cells, a multiplicity of activating receptors can induce signals for adhesion. Engagement of the integrin leukocyte functional antigen-1 mediates firm adhesion, provides signals for granule polarization and orchestrates the structure of an immunological synapse that facilitates efficient target cell killing. Other activating receptors apart from leukocyte functional antigen-1 signal for lytic granule exocytosis, a process that requires overcoming a threshold for activation of phospholipase C-γ, which in turn induces STIM1- and ORAI1-dependent store-operated Ca 〈 sup 〉 2+ 〈 /sup 〉 entry as well as exocytosis mediated by the SNARE-containing protein syntaxin-11 and regulators thereof. Cytokine and chemokine release follows a different secretory pathway which also requires phospholipase C-γ activation and store-operated Ca 〈 sup 〉 2+ 〈 /sup 〉 entry. Recent studies of human NK cells have provided insights into a hierarchy of effector functions that result in graded responses by NK cell populations. Responses display cellular heterogeneity and are influenced by environmental cues. This review highlights recent knowledge gained on the molecular pathways for and regulation of NK cell activation.

Abstract: Maturation of human natural killer (NK) cells as defined by accumulation of cell-surface expression of CD57 is associated with increased cytotoxic character and TNF and IFNγ production upon target-cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacologic inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK-cell maturation including T-BET, ZEB2, and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFNγ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity. In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy. Cancer Res; 77(20); 5664–75. ©2017 AACR.

Abstract: Epigenetic landscapes can provide insight into regulation of gene expression and cellular diversity. Here, we examined the transcriptional and epigenetic profiles of seven human blood natural killer (NK) cell populations, including adaptive NK cells. The BCL11B gene, encoding a transcription factor (TF) essential for T cell development and function, was the most extensively regulated, with expression increasing throughout NK cell differentiation. Several Bcl11b-regulated genes associated with T cell signaling were specifically expressed in adaptive NK cell subsets. Regulatory networks revealed reciprocal regulation at distinct stages of NK cell differentiation, with Bcl11b repressing RUNX2 and ZBTB16 in canonical and adaptive NK cells, respectively. A critical role for Bcl11b in driving NK cell differentiation was corroborated in BCL11B -mutated patients and by ectopic Bcl11b expression. Moreover, Bcl11b was required for adaptive NK cell responses in a murine cytomegalovirus model, supporting expansion of these cells. Together, we define the TF regulatory circuitry of human NK cells and uncover a critical role for Bcl11b in promoting NK cell differentiation and function.

Abstract: The Ikaros family transcription factors regulate lymphocyte development. Loss-of-function variants in IKZF1 cause primary immunodeficiency, but Ikaros family members IKZF2 and IKZF3 have not yet been associated with immunodeficiency. Here, we describe a pedigree with a heterozygous truncating variant in IKZF2 , encoding the transcriptional activator and repressor Helios, which is highly expressed in regulatory T cells and effector T cells, particularly of the CD8 + T cell lineage. Protein-protein interaction analysis revealed that the variant abolished heterodimerization of Helios with Ikaros and Aiolos and also prevented Helios binding to members of the Mi-2/NuRD chromatin remodeling complex. Patients carrying the IKZF2 variant presented with a combined immunodeficiency phenotype characterized by recurrent upper respiratory infections, thrush and mucosal ulcers, and chronic lymphadenopathy. With extensive immunophenotyping, functional assays, and transcriptional analysis, we show that reduced Helios expression was associated with chronic T cell activation and increased production of proinflammatory cytokines both in effector and regulatory T cells. Lymph node histology from patients indicated dysregulated germinal center reactions. Moreover, affected individuals displayed a profound reduction in circulating MAIT cell numbers. In summary, we show that this previously undescribed loss-of-function variant in Helios leads to an immunodeficiency with signs of immune overactivation.

Abstract: Background: Tyrosine kinase inhibitors (TKIs) have significantly improved the treatment of CML. Even though TKI treatment is generally not considered curative, recent studies have shown that nearly half of CML patients who have achieve good and durable responses are able to stop the TKI treatment. However, patients who have successfully discontinued TKI treatment still have residual disease. We hypothesized that the immune system plays a role in treatment free remission (TFR), and our preliminary results in the EURO-SKI trial showed that patients who relapse early after imatinib discontinuation have decreased numbers and frequencies of NK cells. In EURO-SKI trial relapse was defined as the loss of major molecular response (MMR). We now aimed to analyze in more detail the phenotype and function of the NK cells in order to understand their role in TFR. Methods: Lymphocyte subclass analysis (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1 month after the imatinib discontinuation in patients participating in the EURO-SKI stopping trial in the Nordic countries (n=105, results are presented from patients who have reached 6 months follow-up). More detailed immune phenotype and functional assays (NK-cell degranulation and secretion of Th1 type of cytokines IFN-γ/TNF-α) were analyzed from a proportion of patients (n=31). Results: Imatinib treated patients remaining in remission for 6 months (non-relapsing, n=48, median age 60,5 years) displayed an increased amount of NK cells at the time of drug discontinuation (18.6% vs. 11.0%, p=0.02, NK-cell count 0.25 x109 cells/L vs. 0.184 x109 cells/L m, p=0.059) compared to patients who relapsed early (before 5 months, n=29, median age 60,5 years). Furthermore, the NK cell frequency in non-relapsing patients was even higher than in healthy controls (11.5%, n=48, p=0.001). T and B cell counts and frequencies showed no differences between the groups. Detailed analysis of the NK cell compartment displayed a more mature phenotype for the NK cells in non-relapsing patients. Larger frequencies of NK cells from early relapsing patients was CD56bright compared to non-relapsing patients (4.8% vs. 2.7% of CD56 NK cells, p=0.04). Furthermore, patients who had higher frequencies of CD56bright NK cells than median had decreased TFR at 6 months (42%) compared to patients with lower frequency (70%, p=0.01). In addition, there was a trend towards more CD57pos (78% (n=21) vs. 66% (n=10), p=0.09) CD56dim NK cells in non-relapsing patients. To further study the mature NK cells in non-relapsing patients, recently identified markers (FceRgneg, PLZFneg, SYKneg, EAT-2neg) for adaptive NK cells were analyzed. Interestingly, there was a trend that non-relapsing patients had higher frequencies of adaptive-like NK cells. For example, non-relapsing patients had more CD56dim NK cells that had down regulated EAT-2 (2.8% (n=6) vs. 1.3% (n=5) of lymphocytes, p=0.03) and more CD56dim NK cells expressing NKG2D (11.2% vs. 2.6% of lymphocytes, p=0.02) and NKp46 (13.6% vs. 3.9% of lymphocytes, p=0.05). Moreover, after imatinib discontinuation the expression of transcription factor Eomes increased in the CD56dim NK cells of the early relapsing group (baseline MFI 2045 vs. 1 month 3480, p=0.06), while in non-relapsing group it seemed to even decrease (baseline MFI 2273 vs. 1 month 1980, p=0.13) pointing towards an adaptive phenotype. No significant differences between the groups were observed when degranulation against K562 cell line was studied. However, CD16neg NK cells from non-relapsing patients responded to K562 stimulation by secreting more TNFα/IFNγ compared to the early relapsing patients (21% vs. 13% of CD56pos CD16neg NK cells, p=0.01). Furthermore, patients whose CD16neg NK cells had higher than median TNFα/IFNγ secretion when stimulated with K562 cells showed an increased TFR at 6 months (78%) compared to patients who had lower TNFα/IFNγ secretion than median (37%, p=0.005). Conclusions: CML patients who successfully discontinued imatinib therapy displayed a higher number and frequency of peripheral blood mature, adaptive-like NK cells capable of secreting cytokines TNFα/IFNγ relative to relapsing patients. How such NK cells may contribute to maintenance of treatment free remission is still unknown. Nonetheless, our results warrant further clinical studies with NK-cell modulating agents. Disclosures Muller: Novartis: Honoraria, Other: Consulting or Advisory Role, Research Funding; ARIAD Pharmaceuticals Inc.: Honoraria, Other: Consulting & Advisory Role, Research Funding; BMS: Honoraria, Other: Consulting or Advisory Role, Research Funding. Hjorth-Hansen:Novartis: Honoraria; Ariad: Honoraria; Bristol-Myers Squibb: Research Funding; Pfizer: Honoraria, Research Funding. Saussele:Pfizer: Honoraria, Other: Travel grant; BMS: Honoraria, Other: Travel grant, Research Funding; Novartis Pharma: Honoraria, Other: Travel grant, Research Funding; ARIAD: Honoraria. Mahon:ARIAD: Consultancy; Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Richter:Ariad: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria. Mustjoki:the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Academy of Finland: Research Funding; Sigrid Juselius Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Abstract: KIR and NKG2A receptors educate human NK cells to stay responsive to cells with diminished HLA class I. Here, we addressed whether the HLA class I‐binding receptor LIR‐1 (LILRB1/ILT2/CD85j), which is widely expressed on human NK cells, can mediate education and contribute to antitumor functions of NK cells. Methods Healthy donor NK cells either unstimulated, overnight cytokine‐activated or ex vivo ‐expanded were used to target human cell lines. Phenotype and function were analysed using flow cytometry and 51 Cr‐release assays. Results We found that the inhibitory receptor LIR‐1 can mediate NK cell education under specific conditions. This novel finding was exclusive to expanded NK cells and further characterisation of the cells revealed high expression of granzyme B and DNAM‐1, which both previously have been linked to NK cell education. Corroborating the rheostat education model, LIR‐1 co‐expression with an educating KIR further increased the responsiveness of expanded NK cells. Inversely, antibody masking of LIR‐1 decreased the responsiveness. LIR‐1 + expanded NK cells displayed high intrinsic ADCC that, in contrast to KIR and NKG2A, was not inhibited by HLA class I. Conclusion These findings identify a unique NK cell subset attractive for adoptive cell therapy to treat cancer. Given that LIR‐1 binds most HLA class I molecules, this subset may be explored in both autologous and allogeneic settings to innately reject HLA class I ‐ tumor cells as well as HLA class I + target cells when combined with antitumor antibodies. Further studies are warranted to address the potential of this subset in vivo .