Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity

Natural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity.     

Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering.

Natural killer (NK) cell cytotoxicity is determined by a balance of positive and negative signals. Negative signals are transmitted by NK inhibitory receptors (killer immunoglobulin-like receptors, KIR) at the site of membrane apposition between an NK cell and a target cell, where inhibitory receptors become clustered with class I MHC ligands in an organized structure known as an inhibitory NK immune synapse. Immune synapse formation in NK cells is poorly understood. Because signaling by NK inhibitory receptors could be involved in this process, the human NK tumor line YTS was transfected with signal-competent and signal-incompetent KIR2DL1. The latter were generated by truncating the KIR2DL1 cytoplasmic tail or by introducing mutations in the immunoreceptor tyrosine-based inhibition motifs. The KIR2DL1 mutants retained their ability to cluster class I MHC ligands on NK cell interaction with appropriate target cells. Therefore, receptor-ligand clustering at the inhibitory NK immune synapse occurs independently of KIR2DL1 signal transduction. However, parallel examination of NK cell membrane lipid rafts revealed that KIR2DL1 signaling is critical for blocking lipid raft polarization and NK cell cytotoxicity. Moreover, raft polarization was inhibited by reagents that disrupt microtubules and actin filaments, whereas synapse formation was not. Thus, NK lipid raft polarization and inhibitory NK immune synapse formation occur by fundamentally distinct mechanisms.     

CD28-stimulated ERK2 phosphorylation is required for polarization of the microtubule organizing center and granules in YTS NK cells

Activation of natural killer (NK) cell cytotoxicity requires adhesion and formation of a conjugate with a susceptible target cell, followed by actin polymerization, and polarization of the microtubule organizing center (MTOC) and cytolytic granules to the NK cell immune synapse. Here, by using the YTS NK cell line as a model, CD28 is shown to be an activating receptor. It signals cytotoxicity in a process dependent on phosphoinositide-3 kinase activation, leading to sustained extracellular signal-regulated kinase 2 (ERK2) phosphorylation. ERK and phospho-ERK localize to microtubule filaments. Neither conjugation with targets nor actin polymerization is affected by blocking ERK2 activation. However, both polarization of the MTOC and cytolytic granules to the synaptic region and NK cell cytotoxicity are strongly reduced by blocking ERK2 activation. A role for the CD28/CD80 interaction in cytotoxicity of human peripheral NK cells also was established. By contrast, lymphocyte function-associated antigen 1 (LFA-1) ligation transduces only a transient ERK2 activation and fails to induce killing in YTS cells. Thus, in YTS cells, a CD28 signal is used to polarize the MTOC and cytolytic granules to the NK cell immune synapse by stimulating sustained ERK2 activation.     

Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect

In this study we show that Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton that belongs to the Scar/WAVE family, plays a crucial role in the control of natural killer (NK) cell cytotoxicity. Analysis of NK cell numbers and cytotoxic activity in patients carrying different mutations in the WASP coding gene indicated that although the percentage of NK cells was normal or increased, natural cytotoxicity and antibody-mediated NK cell cytotoxicity were inhibited in all patients with the classical WAS phenotype and in most patients carrying mutations associated with the X-linked thrombocytopenia (XLT) phenotype. The inhibition of NK cell-mediated cytotoxicity was associated with the reduced ability of WAS and XLT NK cells to form conjugates with susceptible target cells and to accumulate F-actin on binding. Treatment with interleukin-2 (IL-2) corrected the functional defects of NK cells by affecting their ability to bind to sensitive target cells and to accumulate F-actin. In addition, we provide information on the molecular mechanisms that control WASp function, demonstrating that binding of NK cells to sensitive targets or triggering through CD16 by means of reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly activates Cdc42. We also found that WASp undergoes tyrosine phosphorylation upon CD16 or beta2-integrin engagement on NK cells.     

Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses

The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.     

JNK MAP kinase activation is required for MTOC and granule polarization in NKG2D-mediated NK cell cytotoxicity

Interaction of the activating receptor NKG2D with its ligands is a major stimulatory pathway for cytotoxicity of natural killer (NK) cells. Here, the signaling pathway involved after NKG2D ligation is examined. Either incubation of the NKG2D-bearing human NKL tumor cell line with K562 target cells or cross-linking with NKG2D mAb induced strong activation of the mitogen-activated protein (MAP) kinases. Selective inhibition of JNK MAP kinase with four different means of inhibition greatly reduced NKG2D-mediated cytotoxicity toward target cells and furthermore, blocked the movement of the microtubule organizing center (MTOC), granzyme B (a component of cytotoxic granules), and paxillin (a scaffold protein) to the immune synapse. NKG2D-induced activation of JNK kinase was also blocked by inhibitors of Src protein tyrosine kinases and phospholipase PLCgamma, upstream of JNK. Similarly, a second MAP kinase pathway through ERK was previously shown to be required for NK cell cytotoxicity. Thus, activation of two MAP kinase pathways is required for cytotoxic granule and MTOC polarization and for cytotoxicity of human NK cells when NKG2D is ligated.     

Terminal transport of lytic granules to the immune synapse is mediated by the kinesin-1/Slp3/Rab27a complex

Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1-dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.     

Transfer of NKG2D and MICB at the cytotoxic NK cell immune synapse correlates with a reduction in NK cell cytotoxic function

Although transfer of membrane proteins has been shown to occur during immune cell interactions, the functional significance of this process is not well understood. Here we describe the intercellular transfer of NKG2D and MHC class I chain-related molecule (MIC) B proteins at the cytotoxic natural killer cell immune synapse (cNK-IS). MICB expressed on the 721.221 cell line induced clustering of NKG2D at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing F-actin. Moreover, natural killer (NK) cell membrane-connective structures formed during cytotoxic interactions contained F-actin, perforin, and NKG2D. NKG2D transfer depended on binding to MICB and was specific because transfer of other molecules not involved in NK-IS formation was not observed. Transfer of MICB to NK cells also was noted, suggesting a bidirectional exchange of receptor/ligand pairs at cNK-IS. Experiments designed to test the functional significance of these observations revealed that brief interactions between NK cells and MICB expressing target cells led to a reduction in NKG2D-dependent NK cytotoxicity. These data demonstrate interchange of an activating receptor and its ligand at the cNK-IS and document a correlation between synapse organization, intercellular protein transfer, and compromised NK cell function after interaction with a susceptible target cell.     

Identification of an evolutionarily conserved, function-associated molecule on human natural killer cells.

Nonspecific cytotoxic cells (NCC) in teleost fish are analogous to human natural killer (NK) cells and spontaneously lyse a variety of transformed human cell lines sensitive to human NK cell lysis. Monoclonal antibodies (mAbs) made against these NK-like effector cells inhibited their lytic activity. These anti-NCC mAbs were examined with human NK cells for their effects. It was observed that these mAbs bound specifically to a small percentage of peripheral blood lymphocytes (5-15%) and to the majority (greater than 85%) of CD3- NK cells. The mAbs inhibited human NK cell lysis against a variety of transformed cell lines. Single-cell assays showed that the mAbs exerted their effects through inhibition of conjugate formation (recognition). However, the mAbs did not inhibit NK cell-mediated antibody-dependent cell-mediated cytotoxicity. Biochemical analysis of the NK cell molecule revealed that a dimeric structure was involved (distinct from antigen receptors on T cells). Thus, this molecule appears to be a candidate NK cell antigen receptor.     

Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold

Activation of biological functions in T lymphocytes is determined by the molecular dynamics occurring at the T cell/opposing cell interface. In the present study, a central question of cytotoxic T lymphocyte (CTL) biology was studied at the single-cell level: can two distinct activation thresholds for cytotoxicity and cytokine production be explained by intercellular molecular dynamics between CTLs and targets? In this study, we combine morphological approaches with numerical analysis, which allows us to associate specific patterns of calcium mobilization with different biological responses. We show that CTLs selectively activated to cytotoxicity lack a mature immunological synapse while exhibiting a low threshold polarized secretion of lytic granules and spike-like patterns of calcium mobilization. This finding is contrasted by fully activated CTLs, which exhibit a mature immunological synapse and smooth and sustained calcium mobilization. Our results indicate that intercellular molecular dynamics and signaling characteristics allow the definition of two activation thresholds in individual CTLs: one for polarized granule secretion (lytic synapse formation) and the other for cytokine production (stimulatory synapse formation).     

Activation of natural killer cells with interleukin 2 (IL-2) and IL-12 increases perforin binding and subsequent lysis of tumour cells

Natural killer (NK) cells can lyse a variety of different tumour cells by exocytosis of perforin, subsequent binding of perforin to the target cell membrane and formation of lytic pores. Some tumour cells, however, are resistant to cellular cytotoxicity. Using the NK-resistant tumour cell lines ML-2, MONOMAC-1, RPMI and L540Cy, we demonstrated that activation of NK cells with interleukin 2 (IL-2) and IL-12 resulted in significant lysis of these tumour targets. To investigate the underlying mechanisms, we isolated the cytotoxic granules from non-activated and IL-2-/IL-12-activated NK cells and compared the killing of K562 leukaemia cells (sensitive to NK cell-mediated lysis) and ML-2 leukaemia cells (resistant to NK cell-mediated lysis). In contrast to K562 cells, which were easily killed by NK-cell granules, ML-2 cells were resistant to granules from non-activated NK cells. However, granules from NK cells activated with IL-2 and IL-12 were able to induce significant tumour cell lysis. Cell death of both K562 and ML-2 cells by granules from activated NK cells was completely blocked by anti-perforin antibodies, indicating that perforin mainly accounts for the lysis induced by NK granules. Comparing granules from non-activated and IL-2-/IL-12-activated NK cells, the increased cell death of ML-2 cells was caused by an improved binding of perforin to the target cell membrane. Functional assays, however, indicated that the differences in perforin binding were not as a result of an augmented production of perforin by activated NK cells. We conclude that activation of NK cells results in an increased binding of perforin and subsequent lysis of tumour cells.     

The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells

The ability of natural killer (NK) cells to kill malignant or infected cells depends on the integration of signals from different families of cell surface receptors, including cytokine receptors. How such signals then regulate NK-cell cytotoxicity is incompletely understood. Here we analyzed an endogenous inhibitor of protein phosphatase 2A (PP2A) activity called SET, and its role in regulating human NK-cell cytotoxicity and its mechanism of action in human NK cells. RNAi-mediated suppression of SET down-modulates NK-cell cytotoxicity, whereas ectopic overexpression of SET enhances cytotoxicity. SET knockdown inhibits both mRNA and protein granzyme B expression, as well as perforin expression, whereas SET overexpression enhances granzyme B expression. Treatment of NK cells with the PP2A activator 1,9-dideoxy-forskolin also inhibits both granzyme B expression and cytotoxicity. In addition, pretreatment with the PP2A inhibitor okadaic acid rescues declining granzyme B mRNA levels in SET knockdown cells. Down-modulation of SET expression or activation of PP2A also decreases human NK-cell antibody-dependent cellular cytotoxicity. Finally, the induction of granzyme B gene expression by interleukin-2 and interleukin-15 is inhibited by SET knockdown. These data provide evidence that granzyme B gene expression and therefore human NK-cell cytotoxicity can be regulated by the PP2A-SET interplay.     

Impaired binding of perforin on the surface of tumor cells is a cause of target cell resistance against cytotoxic effector cells

Exocytosis of perforin, subsequent binding of perforin to the target cell membrane, and formation of lytic pores form an important pathway involved in the induction of tumor cell death by cytotoxic effector cells. Here we describe a novel escape mechanism employed by tumor cells to protect themselves from granule-mediated cell death: We were able to demonstrate that the resistance of the human leukemia cell line ML-2 to natural killer (NK)-cell-mediated killing is not caused by impaired NK-cell activation but by resistance against effector molecules contained in the granules of cytotoxic cells. No resistance was observed against other pore-forming agents like complement and streptolysin O. By using the NK-susceptible leukemia cell line K562, we could show that the induction of cell death by cytotoxic granules can be blocked completely by anti-perforin antibodies, indicating that perforin is essentially involved in this process. Flow cytometric data revealed that an impaired binding of perforin on the tumor cell membrane is mainly responsible for target cell resistance, because perforin turned out to bind well on K562 cells but is not able to attach to the surface of ML-2 cells. After impaired binding of perforin was identified as a potential mechanism of tumor cell resistance, leukemia cells from 6 patients with acute myeloid leukemia (AML) were examined. As predicted, AML cells that failed to bind perforin on their surface demonstrated complete resistance toward NK-cell-mediated cytotoxicity. Thus, perforin resistance could represent an important tumor escape mechanism that should be considered when cytotoxic effector cells are used for cellular immunotherapy. (Blood. 2000;96:594-600)     

Radiotherapy transiently reduces the sensitivity of cancer cells to lymphocyte cytotoxicity

The impact of radiotherapy on the interaction between immune cells and cancer cells is important not least because radiotherapy can be used alongside immunotherapy as a cancer treatment. Unexpectedly, we found that X-ray irradiation of cancer cells induced significant resistance to natural killer (NK) cell killing. This was true across a wide variety of cancer-cell types as well as for antibody-dependent cellular cytotoxicity. Resistance appeared 72 h postirradiation and persisted for 2 wk. Resistance could also occur independently of radiotherapy through pharmacologically induced cell-cycle arrest. Crucially, multiple steps in NK-cell engagement, synapse assembly, and activation were unaffected by target cell irradiation. Instead, radiotherapy caused profound resistance to perforin-induced calcium flux and lysis. Resistance also occurred to a structurally similar bacterial toxin, streptolysin O. Radiotherapy did not affect the binding of pore-forming proteins at the cell surface or membrane repair. Rather, irradiation instigated a defect in functional pore formation, consistent with phosphatidylserine-mediated perforin inhibition. In vivo, radiotherapy also led to a significant reduction in NK cell–mediated clearance of cancer cells. Radiotherapy-induced resistance to perforin also constrained chimeric antigen receptor T-cell cytotoxicity. Together, these data establish a treatment-induced resistance to lymphocyte cytotoxicity that is important to consider in the design of radiotherapy–immunotherapy protocols.

Radiotherapy is one of the most commonly used treatments for solid tumors, with approximately half of patients receiving radiotherapy as part of their treatment (1). The classical model for radiotherapy-induced tumor regression involves the production of DNA damage leading to cell-cycle arrest and cell death (2). However, it is now widely recognized that radiotherapy also alters the antitumor immune response in a manner that can critically affect the outcome of treatment (3). Understanding how irradiation affects antitumor immunity by T cells, dendritic cells, and macrophages has driven the development of new immunotherapies that enhance the efficacy of radiotherapy (4). In contrast, little is known about how radiotherapy affects the interaction between natural killer (NK) cells and cancer cells.NK cells, like cytotoxic T cells, contribute to tumor control by secreting cytotoxic granules containing perforin and granzyme B or by expressing ligands, such as TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand, that bind death receptors on target cells (5–7). Secretion of these cytotoxic effector proteins occurs following formation of a lytic immune synapse (8, 9). NK cells polarize the microtubule organizing center (MTOC) and cytotoxic granules toward the immune synapse and then release granule contents into the synaptic cleft in order to kill the target cell (10). Subsequent detachment permits NK cells to carry out multiple sequential kills (11, 12). Novel immunotherapies that harness the effector functions of NK cells have recently been developed for the treatment of cancer (13). However, more must be understood about how radiotherapy affects NK cell–cancer cell interactions if these treatments are to be used effectively in combination.Cancer radiotherapy has been reported to modulate a number of factors which, at least in principle, could influence NK-cell activity. In particular, radiotherapy could up-regulate the expression of stress-inducible activating ligands on cancer cells, which would be expected to increase NK cell–mediated killing (14–18). However, the dynamics of the interaction between NK cells and irradiated cells have not been well studied. Therefore, we set out to test the effect of cancer-cell irradiation on the ability of NK cells to interact with and kill cancer cells. We found that, contrary to what was expected, irradiation of cancer cells resulted in a reduced susceptibility to NK-cell killing. We determined that this was caused by an increased resistance to perforin. This also led to reduced killing by cytotoxic T cells. Thus, these data have broad implications for cancer therapies, especially for combinations of radiotherapy and immunotherapy.     

Fibrin formation inhibits the in vitro cytotoxic activity of human natural and lymphokine-activated killer cells

The biological significance of the interaction of the haemostatic factors with tumour cells remains unclear. It has been hypothesized that fibrin deposition around tumour cells could help those cells to escape destruction by cytotoxic effector cells. To obtain direct evidence in support of this possibility, the effect of fibrin formation on in vitro cytotoxicity of human natural killer (NK) or lymphokine-activated killer (LAK) cells was investigated by comparing their cytotoxic activity with various human tumour cell lines in the presence of human serum or plasma. The data demonstrate that pre-incubation of human tumour cells with plasma, but not serum, substantially diminished or completely abrogated the cytotoxic effects of these killer cells. This was shown to be due to fibrin formation. The degree of coagulation and the number of radioactive tumour cells trapped in the clot correlated with the extent of inhibition of NK or LAK cytotoxicity. Abrogation of LAK activity was also observed when the effector cells were pre-exposed to plasma or when effector and target cells were simultaneously mixed with plasma and trapped in a fibrin clot. Similar results were obtained when, instead of whole plasma, the cytotoxic effect of LAK and NK cells was studied in the presence of fibrinogen and thrombin. When heparin was added, fibrin formation was prevented and no inhibition of LAK/NK cell cytotoxicity was observed. In studies of the mechanisms of inhibition of LAK cell activity by fibrin, target - effector cell conjugate formation was found to be blocked. When plasma was added post-binding (15-30 min after mixing effector and target cells) although coagulation occurred, no effect on cytotoxicity was observed, supporting the conclusion that fibrin interfered with binding rather than the lytic phase of cytotoxic cell activity. Thus, the present data demonstrate that fibrin deposition around tumour and/or effector cells can protect tumour cells from immune destruction and diminish the efficiency of the cytotoxic LAK/NK cells.     

What are killer cells and what do they do?

Cytotoxic effector function of the immune system is mediated predominantly by killer cells and with the exception of complement-mediated lysis all forms of immune-directed cell death are attributable to these cells. The heterogeneous collection of cells with cytotoxic properties ranges from the phylogenetically primitive granulocyte and natural killer (NK) cell lineages to the more complex and versatile macrophage and antigen-specific cytotoxic T lymphocyte. Most killer cells are capable of the dual functions of cytotoxicity and immunoregulation. Granulocytes, macrophages and NK cells constitute an effective 'first line' cellular defence against invasive microbial pathogens and emerging malignancies. Through direct cytotoxicity or by the secretion of cytokines, NK cells also prevent overproliferation of precursor cell populations, thereby exerting a more discriminating control over antigen-specific T and B cell responses. The ultimate refinement of the killer cell is the cytotoxic T cell which is directed by an antigen receptor comparable in specificity and diversity to the immunoglobulin molecule. The mechanisms underlying the initial interaction between killer cell and target and the subsequent lytic event are imperfectly understood. Although many cytokines with lytic properties have been identified and in some cases cloned, their relative importance and intricate interactions with other components of the immune system are still largely unknown. In addition to the prevention of infection and malignancy, killer cells of all lineages are important in the pathogenesis of human disease. Of particular interest is the role of macrophages, NK cells and cytotoxic T cells in autoimmunity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human decidual NK cells form immature activating synapses and are not cytotoxic

In early pregnancy invading fetal trophoblasts encounter abundant maternal decidual natural killer cells (dNK). dNK express perforin, granzymes A and B and the activating receptors NKp30, NKp44, NKp46, NKG2D, and 2B4 as well as LFA-1. Even though they are granular and express the essential molecules required for lysis, fresh dNK displayed very reduced lytic activity on classical MHC I negative targets K562 and 721.221, approximately 15% of that of peripheral NK cells. dNK formed conjugates and activating immune synapses with 721.221 and K562 cells in which CD2, LFA-1 and actin were polarized toward the contact site. However, in contrast to peripheral NK cells, they failed to polarize their microtubule organizing centers and perforin-containing granules to the synapse, accounting for their lack of cytotoxicity.     

Spontaneous and interferon-induced natural cytotoxicity in Crohn's disease

Natural killer cell (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) exerted by peripheral blood mononuclear cells (PMNC) were investigated in 53 patients with Crohn's disease (CD). NK activity and ADCC were found to be significantly reduced in patients with CD (p less than 0.0005) as compared to healthy controls. Both effector cell functions increased after in vitro treatment of PMNC with gamma-interferon, but did not reach the levels found in controls (p less than 0.0005). Neither NK activity nor ADCC was significantly influenced by therapy with corticosteroids. Moreover, the reduced serum zinc levels in patients with CD, which have been shown to be associated with impaired immune function, did not influence the lytic effector cell mechanism assayed either. Finally, no association could be found between NK cell activity or ADCC and CD activity index, the extent of the disease and several laboratory parameters of inflammation. We conclude that patients with CD have a reduced lytic effector cell function which remains uninfluenced by corticosteroid treatment and seems to be present independently of disease activity.     

Activity of lenalidomide in mantle cell lymphoma can be explained by NK cell‐mediated cytotoxicity

Lenalidomide is an immunomodulatory agent that has demonstrated clinical benefit for patients with relapsed or refractory mantle cell lymphoma (MCL); however, despite this observed clinical activity, the mechanism of action (MOA) of lenalidomide has not been characterized in this setting. We investigated the MOA of lenalidomide in clinical samples from patients enrolled in the CC‐5013‐MCL‐002 trial (NCT00875667) comparing single‐agent lenalidomide versus investigator's choice single‐agent therapy and validated our findings in pre‐clinical models of MCL. Our results revealed a significant increase in natural killer (NK) cells relative to total lymphocytes in lenalidomide responders compared to non‐responders that was associated with a trend towards prolonged progression‐free survival and overall survival. Clinical response to lenalidomide was independent of baseline tumour microenvironment expression of its molecular target, cereblon, as well as genetic mutations reported to impact clinical response to the Bruton tyrosine kinase inhibitor ibrutinib. Preclinical experiments revealed lenalidomide enhanced NK cell‐mediated cytotoxicity against MCL cells via increased lytic immunological synapse formation and secretion of granzyme B. In contrast, lenalidomide exhibited minimal direct cytotoxic effects against MCL cells. Taken together, these data provide the first insight into the clinical activity of lenalidomide against MCL, revealing a predominately immune‐mediated MOA.