The transcriptional landscape of αß T cell differentiation.

The differentiation of αßT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

Identification of transcriptional regulators in the mouse immune system.

The differentiation of hematopoietic stem cells into cells of the immune system has been studied extensively in mammals, but the transcriptional circuitry that controls it is still only partially understood. Here, the Immunological Genome Project gene-expression profiles across mouse immune lineages allowed us to systematically analyze these circuits. To analyze this data set we developed Ontogenet, an algorithm for reconstructing lineage-specific regulation from gene-expression profiles across lineages. Using Ontogenet, we found differentiation stage-specific regulators of mouse hematopoiesis and identified many known hematopoietic regulators and 175 previously unknown candidate regulators, as well as their target genes and the cell types in which they act. Among the previously unknown regulators, we emphasize the role of ETV5 in the differentiation of γδ T cells. As the transcriptional programs of human and mouse cells are highly conserved, it is likely that many lessons learned from the mouse model apply to humans.

Molecular definition of the identity and activation of natural killer cells.

Using whole-genome microarray data sets of the Immunological Genome Project, we demonstrate a closer transcriptional relationship between NK cells and T cells than between any other leukocytes, distinguished by their shared expression of genes encoding molecules with similar signaling functions. Whereas resting NK cells are known to share expression of a few genes with cytotoxic CD8(+) T cells, our transcriptome-wide analysis demonstrates that the commonalities extend to hundreds of genes, many encoding molecules with unknown functions. Resting NK cells demonstrate a 'preprimed' state compared with naive T cells, which allows NK cells to respond more rapidly to viral infection. Collectively, our data provide a global context for known and previously unknown molecular aspects of NK cell identity and function by delineating the genome-wide repertoire of gene expression of NK cells in various states.

The efficacy of combination treatment with elotuzumab and lenalidomide is dependent on crosstalk between natural killer cells, monocytes and myeloma cells.

Patients with refractory relapsed multiple myeloma respond to combination treatment with elotuzumab and lenalidomide. The mechanisms underlying this observation are not fully understood. Furthermore, biomarkers predictive of response have not been identified to date. To address these issues, we used a humanized myeloma mouse model and adoptive transfer of human natural killer (NK) cells to show that elotuzumab and lenalidomide treatment controlled myeloma growth, and this was mediated through CD16 on NK cells. In co-culture studies, we showed that peripheral blood mononuclear cells from a subset of patients with refractory relapsed multiple myeloma were effective killers of OPM2 myeloma cells when treated with elotuzumab and lenalidomide, and this was associated with significantly increased expression of CD54 on OPM2 cells. Furthermore, elotuzumab- and lenalidomide-induced OPM2 cell killing and increased OPM2 CD54 expression were dependent on both monocytes and NK cells, and these effects were not mediated by soluble factors alone. At the transcript level, elotuzumab and lenalidomide treatment significantly increased OPM2 myeloma cell expression of genes for trafficking and adhesion molecules, NK cell activation ligands and antigen presentation molecules. In conclusion, our findings suggest that multiple myeloma patients require elotuzumab- and lenalidomide-mediated upregulation of CD54 on autologous myeloma cells, in combination with NK cells and monocytes to mediate an effective anti-tumor response. Furthermore, our data suggest that increased myeloma cell CD54 expression levels could be a powerful predictive biomarker for response to elotuzumab and lenalidomide treatment.

MicroRNA function in NK-cell biology.

The important role of microRNAs in directing immune responses has become increasingly clear. Here, we highlight discoveries uncovering the role of specific microRNAs in regulating the development and function of natural killer (NK) cells. Furthermore, we discuss the impact of NK cells on the entire immune system during global and specific microRNA ablation in the settings of inflammation, infection, and immune dysregulation.

Stage-specific regulation of natural killer cell homeostasis and response against viral infection by microRNA-155.

Natural killer (NK) cells function in the recognition and destruction of host cells infected with pathogens. Many regulatory mechanisms govern the potent responses of NK cells, both at the cellular and molecular level. Ablation of microRNA (miRNA) processing enzymes demonstrated that miRNAs play critical roles in NK cell differentiation and function; however, the role of individual miRNAs requires further investigation. Using mice containing a targeted deletion of microRNA-155 (miR-155), we observed defects in NK cell maintenance and maturation at steady state, as well as in homeostatic proliferation in lymphopenic mice. In addition, we discovered that miR-155 is up-regulated in activated NK cells during mouse cytomegalovirus (MCMV) infection in response to signals from the proinflammatory cytokines IL-12 and IL-18 and through signal transducer and activator of transcription 4 (STAT4) signaling. Although miR-155 was found to be dispensable for cytotoxicity and cytokine production when triggered through activating receptors, NK cells lacking miR-155 exhibited severely impaired effector and memory cell numbers in both lymphoid and nonlymphoid tissues after MCMV infection. We demonstrate that miR-155 differentially targets Noxa and suppressor of cytokine signaling 1 (SOCS1) in NK cells at distinct stages of homeostasis and activation. NK cells constitutively expressing Noxa and SOCS1 exhibit profound defects in expansion during the response to MCMV infection, suggesting that their regulation by miR-155 promotes antiviral immunity.

Mislocalization of SLP-76 leads to aberrant inflammatory cytokine and autoantibody production.

Central and peripheral tolerance is required to prevent immune responses to self-antigens. We now present a mouse model in which wild-type (WT) SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) has been constitutively targeted to the membrane, where CD4+ T cells become spontaneously dysregulated and develop an inflammatory phenotype. Mice bearing membrane-targeted SLP-76 (MTS) have a partial T-cell lymphopenia and impaired signaling though the mature T-cell receptor. The CD4+ T cells that develop in these mice possess an activated-like phenotype and are skewed toward the inflammatory T(H)1 and T(H)17 lineages. MTS mice also spontaneously develop autoantibodies at an early age. To rule out abnormal thymic selection as the sole cause of the MTS phenotype, we expressed WT SLP-76 along with the MTS followed by deletion of the WT allele in peripheral T cells. The peripheral MTS-expressing T cells demonstrate skewed cytokine responses when transferred into lymphopenic hosts. Thus, the abnormal effector T-cell phenotype still occurs in the presence of preserved central and peripheral tolerance, suggesting that diminished T-cell receptor signaling can promote skewed T-cell responses.

Distinct requirements of microRNAs in NK cell activation, survival, and function.

MicroRNAs (miRNAs) are small noncoding RNAs that have recently emerged as critical regulators of gene expression within the immune system. In this study, we used mice with conditional deletion of Dicer and DiGeorge syndrome critical region 8 (Dgcr8) to dissect the roles of miRNAs in NK cell activation, survival, and function during viral infection. We developed a system for deletion of either Dicer or Dgcr8 in peripheral NK cells via drug-induced Cre activity. We found that Dicer- and Dgcr8-deficient NK cells were significantly impaired in survival and turnover, and had impaired function of the ITAM-containing activating NK cell receptors. We further demonstrated that both Dicer- and Dgcr8-dependent pathways were indispensable for the expansion of Ly49H(+) NK cells during mouse cytomegalovirus infection. Our data indicate similar phenotypes for Dicer- and Dgcr8-deficient NK cells, which strongly suggest that these processes are regulated by miRNAs. Thus, our findings indicate a critical role for miRNAs in controlling NK cell homeostasis and effector function, with implications for miRNAs regulating diverse aspects of NK cell biology.

Cutting edge: rescue of pre-TCR but not mature TCR signaling in mice expressing membrane-targeted SLP-76.

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) organizes signaling from immunoreceptors, including the platelet collagen receptor, the pre-TCR, and the TCR, and is required for T cell development. In this study we examine a mouse in which wild-type SLP-76 is replaced with a mutant constitutively targeted to the cell membrane. Membrane-targeted SLP-76 (MTS) supports ITAM signaling in platelets and from the pre-TCR. Signaling from the mature TCR, however, is defective in MTS thymocytes, resulting in failed T cell differentiation. Defective thymic selection by MTS is not rescued by a SLP-76 mutant whose localization is restricted to the cytosol. Thus, fixed localization of SLP-76 reveals differential requirements for the subcellular localization of signaling complexes downstream of the pre-TCR vs mature TCR.

Alterations of NK Cell Phenotype in the Disease Course of Multiple Myeloma.

Accumulating evidence demonstrates important roles for natural killer (NK) cells in controlling multiple myeloma (MM). A prospective flow cytometry-based analysis of NK cells in the blood and bone marrow (BM) of MM patient subgroups was performed (smoldering (SMM), newly diagnosed (ND), relapsed/refractory, (RR) and post-stem cell transplantation (pSCT)). Assessments included the biomarker expression and function of NK cells, correlations between the expression of receptors on NK cells with their ligands on myeloma cells, and comparisons between MM patient subgroups and healthy controls. The most striking differences from healthy controls were found in RR and pSCT patients, in which NK cells were less mature and expressed reduced levels of the activating receptors DNAM-1, NKG2D, and CD16. These differences were more pronounced in the BM than in blood, including upregulation of the therapeutic targets TIM3, TIGIT, ICOS, and GITR. Their expression suggests NK cells became exhausted upon chronic encounters with the tumor. A high expression of SLAMF7 on blood NK cells correlated with shorter progression-free survival. This correlation was particularly evident in ND patients, including on mature CD56dim NK cells in the BM. Thus, our NK cell analysis identified possible therapeutic targets in MM and a biomarker with prognostic potential for disease progression.

von Hippel-Lindau mutation in mice recapitulates Chuvash polycythemia via hypoxia-inducible factor-2alpha signaling and splenic erythropoiesis.

The R200W mutation in the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) is unique in that it is not associated with tumor development, but rather with Chuvash polycythemia, a heritable disease characterized by elevated hematocrit and increased serum levels of erythropoietin and VEGF. Previous studies have implicated hypoxia-inducible factor-1alpha (HIF-1alpha) signaling in this disorder, although the effects of this mutation on pVHL function are not fully understood. In order to explore the mechanisms underlying the development of this polycythemia, we generated mice homozygous for the R200W mutation (Vhl(R/R)). Vhl(R/R) mice developed polycythemia highly similar to the human disease. The activity of HIF proteins, specifically the HIF-2alpha isoform, was upregulated in ES cells and tissues from Vhl(R/R) mice. Furthermore, we observed a striking phenotype in Vhl(R/R) spleens, with greater numbers of erythroid progenitors and megakaryocytes and increased erythroid differentiation of Vhl(R/R) splenic cells in vitro. These findings suggest that enhanced expression of key HIF-2alpha genes promotes splenic erythropoiesis, resulting in the development of polycythemia in Vhl(R/R) mice. This mouse model is a faithful recapitulation of this VHL-associated syndrome and represents a useful tool for studying polycythemias and investigating potential therapeutics.

Preclinical Rationale for Targeting the PD-1/PD-L1 Axis in Combination with a CD38 Antibody in Multiple Myeloma and Other CD38-Positive Malignancies.

The CD38-targeting antibody daratumumab mediates its anti-myeloma activities not only through Fc-receptor-dependent effector mechanisms, but also by its effects on T-cell immunity through depletion of CD38+ regulatory T-cells, regulatory B-cells, and myeloid-derived suppressor cells. Therefore, combining daratumumab with modulators of other potent immune inhibitory pathways, such as the PD-1/PD-L1 axis, may further improve its efficacy. We show that multiple myeloma (MM) cells from relapsed/refractory patients have increased expression of PD-L1, compared to newly diagnosed patients. Furthermore, PD-1 is upregulated on T-cells from both newly diagnosed and relapsed/refractory MM patients, compared to healthy controls. In short-term experiments with bone marrow samples from MM patients, daratumumab-mediated lysis was mainly associated with the MM cells' CD38 expression levels and the effector (NK-cells/monocytes/T-cells)-to-target ratio, but not with the PD-L1 expression levels or PD-1+ T-cell frequencies. Although PD-1 blockade with nivolumab did not affect MM cell viability or enhanced daratumumab-mediated lysis in short-term ex vivo experiments, nivolumab resulted in a mild but clear increase in T-cell numbers. Moreover, with a longer treatment duration, PD-1 blockade markedly improved anti-CD38 antibody-mediated cytotoxicity in vivo in murine CD38+ tumor models. In conclusion, dual targeting of CD38 and PD-1 may represent a promising strategy for treating MM and other CD38-positive malignancies.

Enhanced SLAMF7 Homotypic Interactions by Elotuzumab Improves NK Cell Killing of Multiple Myeloma.

Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, and CD319), which is highly expressed on multiple myeloma (MM) cells, natural killer (NK) cells, and subsets of other leukocytes. By engaging with FcγRIIIA (CD16), Elo promotes potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) toward SLAMF7+ MM tumor cells. Relapsed/refractory MM patients treated with the combination of Elo, lenalidomide, and dexamethasone have improved progression-free survival. We previously showed that Elo enhances NK cell activity via a costimulation mechanism, independent of CD16 binding. Here, we further studied the effect of Elo on cytotoxicity of CD16-negative NK-92 cells. Elo, but not other SLAMF7 antibodies, uniquely enhanced cytotoxicity mediated by CD16-negative NK-92 cells toward SLAMF7+ target cells. Furthermore, this CD16-independent enhancement of cytotoxicity required expression of SLAMF7 containing the full cytoplasmic domain in the NK cells, implicating costimulatory signaling. The CD16-independent costimulation by Elo was associated with increased expression of NKG2D, ICAM-1, and activated LFA-1 on NK cells, and enhanced cytotoxicity was partially reduced by NKG2D blocking antibodies. In addition, an Fc mutant form of Elo that cannot bind CD16 promoted cytotoxicity of SLAMF7+ target cells by NK cells from most healthy donors, especially if previously cultured in IL2. We conclude that in addition to promoting NK cell-mediated ADCC (CD16-dependent) responses, Elo promoted SLAMF7-SLAMF7 interactions in a CD16-independent manner to enhance NK cytotoxicity toward MM cells.

Antibody-Dependent Cellular Phagocytosis by Macrophages is a Novel Mechanism of Action of Elotuzumab.

Elotuzumab, a recently approved antibody for the treatment of multiple myeloma, has been shown to stimulate Fcγ receptor (FcγR)-mediated antibody-dependent cellular cytotoxicity by natural killer (NK) cells toward myeloma cells. The modulatory effects of elotuzumab on other effector cells in the tumor microenvironment, however, has not been fully explored. Antibody-dependent cellular phagocytosis (ADCP) is a mechanism by which macrophages contribute to antitumor potency of monoclonal antibodies. Herein, we studied the NK cell independent effect of elotuzumab on tumor-associated macrophages using a xenograft tumor model deficient in NK and adaptive immune cells. We demonstrate significant antitumor efficacy of single-agent elotuzumab in immunocompromised xenograft models of multiple myeloma, which is in part mediated by Fc-FcγR interaction of elotuzumab with macrophages. Elotuzumab is shown in this study to induce phenotypic activation of macrophages in vivo and mediates ADCP of myeloma cells though a FcγR-dependent manner in vitro Together, these findings propose a novel immune-mediated mechanism by which elotuzumab exerts anti-myeloma activity and helps to provide rationale for combination therapies that can enhance macrophage activity. Mol Cancer Ther; 17(7); 1454-63. ©2018 AACR.

The anti-SLAMF7 antibody elotuzumab mediates NK cell activation through both CD16-dependent and -independent mechanisms.

Elotuzumab is a humanized therapeutic monoclonal antibody directed to the surface glycoprotein SLAMF7 (CS1, CRACC, CD319), which is highly expressed on multiple myeloma (MM) tumor cells. Improved clinical outcomes have been observed following treatment of MM patients with elotuzumab in combination with lenalidomide or bortezomib. Previous work showed that elotuzumab stimulates NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), via Fc-domain engagement with FcγRIIIa (CD16). SLAMF7 is also expressed on NK cells, where it can transmit stimulatory signals. We tested whether elotuzumab can directly activate NK cells via ligation with SLAMF7 on NK cells in addition to targeting ADCC through CD16. We show that elotuzumab strongly promoted degranulation and activation of NK cells in a CD16-dependent manner, and a non-fucosylated form of elotuzumab with higher affinity to CD16 exhibited enhanced potency. Using F(ab')2 or Fc-mutant forms of the antibody, the direct binding of elotuzumab to SLAMF7 alone could not stimulate measurable CD69 expression or degranulation of NK cells. However, the addition of soluble elotuzumab could costimulate calcium signaling responses triggered by multimeric engagement of NKp46 and NKG2D in a CD16-independent manner. Thus, while elotuzumab primarily stimulates NK cells through CD16, it can also transduce effective "trans"-costimulatory signals upon direct engagement with SLAMF7, since these responses did not require direct co-engagement with the activating receptors. Trans-costimulation by elotuzumab has potential to reduce activation thresholds of other NK cell receptors engaging with their ligands on myeloma target cell surfaces, thereby potentially further increasing NK cell responsiveness in patients.

PD-1 blockade enhances elotuzumab efficacy in mouse tumor models.

Elotuzumab, a humanized monoclonal antibody that binds human signaling lymphocytic activation molecule F7 (hSLAMF7) on myeloma cells, was developed to treat patients with multiple myeloma (MM). Elotuzumab has a dual mechanism of action that includes the direct activation of natural killer (NK) cells and the induction of NK cell-mediated antibody-dependent cellular cytotoxicity. This study aimed to characterize the effects of elotuzumab on NK cells in vitro and in patients with MM and to determine whether elotuzumab antitumor activity was improved by programmed death receptor-1 (PD-1) blockade. Elotuzumab promoted NK cell activation when added to a coculture of human NK cells and SLAMF7-expressing myeloma cells. An increased frequency of activated NK cells was observed in bone marrow aspirates from elotuzumab-treated patients. In mouse tumor models expressing hSLAMF7, maximal antitumor efficacy of a murine immunoglobulin G2a version of elotuzumab (elotuzumab-g2a) required both Fcγ receptor-expressing NK cells and CD8+ T cells and was significantly enhanced by coadministration of anti-PD-1 antibody. In these mouse models, elotuzumab-g2a and anti-PD-1 combination treatment promoted tumor-infiltrating NK and CD8+ T-cell activation, as well as increased intratumoral cytokine and chemokine release. These observations support the rationale for clinical investigation of elotuzumab/anti-PD-1 combination therapy in patients with MM.

In vitro and in vivo models analyzing von Hippel-Lindau disease-specific mutations.

Mutations in the von Hippel-Lindau (VHL) tumor suppressor gene cause tissue-specific tumors, with a striking genotype-phenotype correlation. Loss of VHL expression predisposes to hemangioblastoma and clear cell renal cell carcinoma, whereas specific point mutations predispose to pheochromocytoma, polycythemia, or combinations of hemangioblastoma, renal cell carcinoma, and/or pheochromocytoma. The VHL protein (pVHL) has been implicated in many cellular activities including the hypoxia response, cell cycle arrest, apoptosis, and extracellular matrix remodeling. We have expressed missense pVHL mutations in Vhl(-/-) murine embryonic stem cells to test genotype-phenotype correlations in euploid cells. We first examined the ability of mutant pVHL to direct degradation of the hypoxia inducible factor (HIF) subunits HIF1alpha and HIF2alpha. All mutant pVHL proteins restored proper hypoxic regulation of HIF1alpha, although one VHL mutation (VHL(R167Q)) displayed impaired binding to Elongin C. This mutation also failed to restore HIF2alpha regulation. In separate assays, these embryonic stem cells were used to generate teratomas in immunocompromised mice, allowing independent assessment of the effects of specific VHL mutations on tumor growth. Surprisingly, teratomas expressing the VHL(Y112H) mutant protein displayed a growth disadvantage, despite restoring HIFalpha regulation. Finally, we observed increased microvessel density in teratomas derived from Vhl(-/-) as well as VHL(Y112H), VHL(R167Q), and VHL(R200W) embryonic stem cells. Together, these observations support the hypothesis that pVHL plays multiple roles in the cell, and that these activities can be separated via discrete VHL point mutations. The ability to dissect specific VHL functions with missense mutations in a euploid model offers a novel opportunity to elucidate the activities of VHL as a tumor suppressor.

Proapoptotic Bim regulates antigen-specific NK cell contraction and the generation of the memory NK cell pool after cytomegalovirus infection.

Apoptosis is critical for the elimination of activated lymphocytes after viral infection. Proapoptotic factor Bim (Bcl2l11) controls T lymphocyte contraction and the formation of memory T cells after infection. Natural killer (NK) cells also undergo antigen-driven expansion to become long-lived memory cells after mouse cytomegalovirus (MCMV) infection; therefore, we examined the role of Bim in regulating the MCMV-driven memory NK cell pool. Despite responding similarly early after infection, Bcl2l11(-/-) Ly49H(+) NK cells show impaired contraction and significantly outnumber wild-type (WT) cells after the expansion phase. The inability to reduce the effector pool leads to a larger Bcl2l11(-/-) NK memory subset, which displays a less mature phenotype (CD11b(lo), CD27(+)) and lower levels of NK cell memory-associated markers KLRG1 and Ly6C. Bcl2l11(-/-) memory NK cells demonstrate a reduced response to m157-mediated stimulation and do not protect as effectively as WT memory NK cells in an MCMV challenge model. Thus, Bim-mediated apoptosis drives selective contraction of effector NK cells to generate a pool of mature, MCMV-specific memory cells.

Proinflammatory cytokine signaling required for the generation of natural killer cell memory.

Although natural killer (NK) cells are classified as innate immune cells, recent studies demonstrate that NK cells can become long-lived memory cells and contribute to secondary immune responses. The precise signals that promote generation of long-lived memory NK cells are unknown. Using cytokine receptor-deficient mice, we show that interleukin-12 (IL-12) is indispensible for mouse cytomegalovirus (MCMV)-specific NK cell expansion and generation of memory NK cells. In contrast to wild-type NK cells that proliferated robustly and resided in lymphoid and nonlymphoid tissues for months after MCMV infection, IL-12 receptor-deficient NK cells failed to expand and were unable to mediate protection after MCMV challenge. We further demonstrate that a STAT4-dependent IFN-γ-independent mechanism contributes toward the generation of memory NK cells during MCMV infection. Understanding the full contribution of inflammatory cytokine signaling to the NK cell response against viral infection will be of interest for the development of vaccines and therapeutics.

miR-150 regulates the development of NK and iNKT cells.

Natural killer (NK) and invariant NK T (iNKT) cells are critical in host defense against pathogens and for the initiation of adaptive immune responses. miRNAs play important roles in NK and iNKT cell development, maturation, and function, but the roles of specific miRNAs are unclear. We show that modulation of miR-150 expression levels has a differential effect on NK and iNKT cell development. Mice with a targeted deletion of miR-150 have an impaired, cell lineage-intrinsic defect in their ability to generate mature NK cells. Conversely, a gain-of-function miR-150 transgene promotes the development of NK cells, which display a more mature phenotype and are more responsive to activation. In contrast, overexpression of miR-150 results in a substantial reduction of iNKT cells in the thymus and in the peripheral lymphoid organs. The transcription factor c-Myb has been shown to be a direct target of miR-150. Our finding of increased NK cell and decreased iNKT cell frequencies in Myb heterozygous bone marrow chimeras suggests that miR-150 differentially controls the development of NK and iNKT cell lineages by targeting c-Myb.