Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor–infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin–mediated TGF-β activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-β signaling or with TGFBR2 gene–edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-β axis as a potentially useful therapeutic target in GBM.     

The SIRPα–CD47 immune checkpoint in NK cells

Here we report on the existence and functionality of the immune checkpoint signal regulatory protein α (SIRPα) in NK cells and describe how it can be modulated for cell therapy. NK cell SIRPα is up-regulated upon IL-2 stimulation, interacts with target cell CD47 in a threshold-dependent manner, and counters other stimulatory signals, including IL-2, CD16, or NKG2D. Elevated expression of CD47 protected K562 tumor cells and mouse and human MHC class I–deficient target cells against SIRPα⁺ primary NK cells, but not against SIRPα⁻ NKL or NK92 cells. SIRPα deficiency or antibody blockade increased the killing capacity of NK cells. Overexpression of rhesus monkey CD47 in human MHC-deficient cells prevented cytotoxicity by rhesus NK cells in a xenogeneic setting. The SIRPα–CD47 axis was found to be highly species specific. Together, the results demonstrate that disruption of the SIRPα–CD47 immune checkpoint may augment NK cell antitumor responses and that elevated expression of CD47 may prevent NK cell–mediated killing of allogeneic and xenogeneic tissues.     

TGF-β signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development

Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-β (TGF-β), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-β inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-β responses, any role of TGF-β in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-β promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant–negative mutant (TβRIIΔcyt) of the TGF-β type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-β. In a murine model of bone metastases, expression of TβRIIΔcyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant–negative signaling blockade by expression of a constitutively active TGF-β type I receptor in the breast cancer cells increased tumor production of parathyroid hormone–related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant–negative TβRIIΔcyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-β in the development of breast cancer metastasis to bone, via the TGF-β receptor–mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.     

CD4+ FoxP3+ regulatory T cells confer infectious tolerance in a TGF-beta-dependent manner

CD4⁺FoxP3⁺ regulatory T (T reg) cells comprise a separate lineage of T cells that are essential for maintaining immunological tolerance to self. The molecular mechanism(s) by which T reg cells mediate their suppressive effects remains poorly understood. One molecule that has been extensively studied in T reg cell suppression is transforming growth factor (TGF)-β, but its importance remains controversial. We found that TGF-β complexed to latency-associated peptide (LAP) is expressed on the cell surface of activated but not resting T reg cells. T reg cell LAP–TGF-β plays an important role in the suppression of the proliferation of activated T cells, but it is not required for the suppression of naive T cell activation. More importantly, T reg cell–derived TGF-β could generate de novo CD4⁺FoxP3⁺ T cells in vitro from naive precursors in a cell contact–dependent, antigen-presenting cell–independent and α_V integrin–independent manner. The newly induced CD4⁺FoxP3⁺ T cells are suppressive both in vitro and in vivo. Transfer of activated antigen-specific T reg cells with naive antigen-specific responder T cells to normal recipients, followed by immunization, also results in induction of FoxP3 expression in the responder cells. T reg cell–mediated generation of functional CD4⁺FoxP3⁺ cells via this TGF-β–dependent pathway may represent a major mechanism as to how T reg cells maintain tolerance and expand their suppressive abilities.     

Cytokines reinstate NK cell–mediated cancer immunosurveillance

In healthy individuals, cells that lose expression of MHC class I molecules are quickly targeted for elimination by NK lymphocytes. A paradox in cancer immunology is the observation that many tumor cells often have a drastic reduction of MHC class I molecules, yet these cells are not eliminated by NK cells, as they should be. In this issue of the JCI, Ardolino et al. demonstrate that NK cells that infiltrate MHC class I–deficient tumors acquire an anergic state that can be reversed by particular combinations of exogenous cytokines. These results indicate that IL-12 plus IL-18 or a recombinant interleukin engineered to stimulate the IL-2 receptor β/γ heterodimer (but not the IL-2 receptor α/β/γ complex) have the potential to be used clinically to reinstate immunosurveillance against MHC class I–deficient tumors.     

Activity and Phenotype of Natural Killer Cells in Peptide Transporter (TAP)-deficient Patients (Type I Bare Lymphocyte Syndrome)

In this paper we describe the function and phenotype of natural killer (NK) lymphocytes from HLA class I–deficient patients. These cells are, as has been previously reported, unable to lyse HLA class I⁻ K562 cells, but are able to perform antibody-dependent cellular cytotoxicity (ADCC), although with lower efficiency as compared to NK cells from normal individuals. Transporter associated to antigen processing (TAP)⁻ NK cells proliferate when cultured in the presence of lymphoblastoid B cells (B-LCs) and interleukin 2 and develop a spectrum of cytotoxicity similar to that of activated normal NK cells. Importantly, activation of the TAP⁻ NK cells induces strong cytotoxicity to autologous B-LCs. Analysis of the phenotype of circulating TAP⁻ NK lymphocytes showed them to display a normal diverse repertoire of HLA class I–specific NK receptors. These receptors were expressed at normal levels, apart from the CD94–NKG2A complex, which appeared to be overexpressed. This latter finding could reflect an adaptation to the low expression of HLA class I molecules. Finally, functional analyses indicated that the inhibitory receptors in TAP⁻ individuals can transduce inhibitory signals. Our results suggest that in vivo, the NK cells of TAP⁻ patients could participate in immune defense, at least through ADCC, but upon activation, may be involved in autoimmune processes.Type I bare lymphocyte syndrome is a rare disease characterized by a strong reduction in the cell surface expression of HLA class I molecules. The patients are not reported to suffer from severe viral infections, which suggests that cell-mediated cytotoxic immune responses are efficient to some extent. However, chronic lung inflammation develops in late childhood. A few years ago we described two siblings, EMO and EFA, who are homozygous for a stop mutation in the gene encoding the TAP2 subunit of the peptide transporter associated to antigen processing (TAP; 1). As a result of this deficiency, most HLA class I molecules remain peptide-free and cannot reach the cell surface. Thus, the TAP-deficient cells from these patients express <3% of HLA class I molecules as compared to normal cells. Nevertheless, CD8⁺ α/β T cells are present among their PBMCs. Recent observations (2) suggest that some of these cells may recognize TAP-independent HLA class I–restricted viral antigens and participate in the development of the immune response, thus explaining the absence of a greater susceptibility to viral infection in these patients.Immune responses are also controlled by NK cells. These lymphocytes are cytotoxic to certain tumor cells, HLA class I⁻ cells, and virus-infected cells and mediate antibody-dependent cellular cytotoxicity (ADCC; 3). The importance of these cells in human immune responses is indicated by the association of severe infections with herpesvirus and EBV with an absence of NK cells or with a reduction of their activity (4, 5).Several molecules expressed at the surface of NK cells are involved in the recognition of target cells and control their cytotoxic activity (6). Some are activating receptors; among these is the type III low affinity receptor for IgG, or CD16, which is involved in ADCC. Other receptors, called killer inhibitory receptors (KIRs), block the cytotoxic process when they interact with the HLA class I molecules expressed on normal cells. In humans, these receptors may be classified in two main families. The receptors specific for subsets of the alleles of HLA-C (p58), HLA-B (p70), and HLA-A (p140) belong to the Ig superfamily (Ig-SF; 6). In contrast, the CD94–NKG2A (or -B) receptor complex is composed of proteins homologous to C-type lectins and displays a broader specificity for different HLA class I alleles (7). More recently, receptors have been described that are homologous to KIRs, but activate the cytotoxicity of NK cells (6, 7).In a previous paper, we reported that NK cells from TAP-deficient patients were unable to lyse HLA class I⁻ K562 tumor cells, suggesting that these cells were not functional (1). However, since the patients do not seem to suffer from severe herpesvirus or EBV infections, this may indicate that their NK cells possess immunological functions. The cytotoxic activity of NK cells from TAP-deficient patients was therefore reexamined in the present study. In a second step, we investigated whether a defective expression of class I molecules could affect the repertoire of HLA class I–specific receptors on NK cells. Finally, we explored the functionality of the inhibitory receptors of these cells.     

The TEL-AML1 leukemia fusion gene dysregulates the TGF-β pathway in early B lineage progenitor cells

Chromosome translocation to generate the TEL-AML1 (also known as ETV6-RUNX1) chimeric fusion gene is a frequent and early or initiating event in childhood acute lymphoblastic leukemia (ALL). Our starting hypothesis was that the TEL-AML1 protein generates and maintains preleukemic clones and that conversion to overt disease requires secondary genetic changes, possibly in the context of abnormal immune responses. Here, we show that a murine B cell progenitor cell line expressing inducible TEL-AML1 proliferates at a slower rate than parent cells but is more resistant to further inhibition of proliferation by TGF-β. This facilitates the competitive expansion of TEL-AML1–expressing cells in the presence of TGF-β. Further analysis indicated that TEL-AML1 binds to a principal TGF-β signaling target, Smad3, and compromises its ability to activate target promoters. In mice expressing a TEL-AML1 transgene, early, pre-pro-B cells were increased in number and also showed reduced sensitivity to TGF-β–mediated inhibition of proliferation. Moreover, expression of TEL-AML1 in human cord blood progenitor cells led to the expansion of a candidate preleukemic stem cell population that had an early B lineage phenotype (CD34⁺CD38^–CD19⁺) and a marked growth advantage in the presence of TGF-β. Collectively, these data suggest a plausible mechanism by which dysregulated immune responses to infection might promote the malignant evolution of TEL-AML1–expressing preleukemic clones.     

CD1d-restricted Recognition of Synthetic Glycolipid Antigens by Human Natural Killer T Cells

A conserved subset of mature circulating T cells in humans expresses an invariant Vα24-JαQ T cell receptor (TCR)-α chain rearrangement and several natural killer (NK) locus–encoded C-type lectins. These human T cells appear to be precise homologues of the subset of NK1.1⁺ TCR-α/β⁺ T cells, often referred to as NK T cells, which was initially identified in mice. Here we show that human NK T cell clones are strongly and specifically activated by the same synthetic glycolipid antigens as have been shown recently to stimulate murine NK T cells. Responses of human NK T cells to these synthetic glycolipids, consisting of certain α-anomeric sugars conjugated to an acylated phytosphingosine base, required presentation by antigen-presenting cells expressing the major histocompatibility complex class I–like CD1d protein. Presentation of synthetic glycolipid antigens to human NK T cells required internalization of the glycolipids by the antigen-presenting cell and normal endosomal targeting of CD1d. Recognition of these compounds by human NK T cells triggered proliferation, cytokine release, and cytotoxic activity. These results demonstrate a striking parallel in the specificity of NK T cells in humans and mice, thus providing further insight into the potential mechanisms of immune recognition by NK T cells and the immunological function of this unique T cell subset.     

Engagement of Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor β (TGF-β) Production by Murine CD4+ T Cells

Evidence indicates that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor β (TGF-β) production by murine CD4⁺ T cells. CD4⁺ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-β after antibody cross-linking of CTLA-4, indicating that induction of TGF-β by CTLA-4 signaling represents a ubiquitous feature of murine CD4⁺ T cells. Stimulation of the CD3–T cell antigen receptor complex does not independently induce TGF-β, but is required for optimal CTLA-4–mediated TGF-β production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon γ (Th1) and IL-4 (Th2). Moreover, addition of anti–TGF-β partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-β1 gene–deleted (TGF-β1^−/−) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4⁺ T cell production of TGF-β, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-β, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4⁺ T cell activation.     

Differential natural killer cell-mediated inhibition of HIV-1 replication based on distinct KIR/HLA subtypes

Decline of peak viremia during acute HIV-1 infection occurs before the development of vigorous adaptive immunity, and the level of decline correlates inversely with the rate of AIDS progression, implicating a potential role for the innate immune response in determining disease outcome. The combined expression of an activating natural killer (NK) cell receptor, the killer immunoglobulin-like receptor (KIR) 3DS1, and its presumed ligand, human leukocyte antigen (HLA)–B Bw4-80I, has been associated in epidemiological studies with a slow progression to AIDS. We examined the functional ability of NK cells to differentially control HIV-1 replication in vitro based on their KIR and HLA types. NK cells expressing KIR3DS1 showed strong, significant dose- and cell contact–dependent inhibition of HIV-1 replication in target cells expressing HLA-B Bw4-80I compared with NK cells that did not express KIR3DS1. Furthermore, KIR3DS1⁺ NK cells and NKLs were preferentially activated, and lysed HIV-1 infected target cells in an HLA-B Bw4-80I–dependent manner. These data provide the first functional evidence that variation at the KIR locus influences the effectiveness of NK cell activity in the containment of viral replication.     

Continuous engagement of a self-specific activation receptor induces NK cell tolerance

Natural killer (NK) cell tolerance mechanisms are incompletely understood. One possibility is that they possess self-specific activation receptors that result in hyporesponsiveness unless modulated by self–major histocompatability complex (MHC)–specific inhibitory receptors. As putative self-specific activation receptors have not been well characterized, we studied a transgenic C57BL/6 mouse that ubiquitously expresses m157 (m157-Tg), which is the murine cytomegalovirus (MCMV)–encoded ligand for the Ly49H NK cell activation receptor. The transgenic mice were more susceptible to MCMV infection and were unable to reject m157-Tg bone marrow, suggesting defects in Ly49H⁺ NK cells. There was a reversible hyporesponsiveness of Ly49H⁺ NK cells that extended to Ly49H-independent stimuli. Continuous Ly49H–m157 interaction was necessary for the functional defects. Interestingly, functional defects occurred when mature wild-type NK cells were adoptively transferred to m157-Tg mice, suggesting that mature NK cells may acquire hyporesponsiveness. Importantly, NK cell tolerance caused by Ly49H–m157 interaction was similar in NK cells regardless of expression of Ly49C, an inhibitory receptor specific for a self-MHC allele in C57BL/6 mice. Thus, engagement of self-specific activation receptors in vivo induces an NK cell tolerance effect that is not affected by self-MHC–specific inhibitory receptors.     

TGF-β induces miR-182 to sustain NF-κB activation in glioma subsets

The strength and duration of NF-κB signaling are tightly controlled by multiple negative feedback mechanisms. However, in cancer cells, these feedback loops are overridden through unclear mechanisms to sustain oncogenic activation of NF-κB signaling. Previously, we demonstrated that overexpression of miR-30e* directly represses IκBα expression and leads to hyperactivation of NF-κB. Here, we report that miR-182 was overexpressed in a different set of gliomas with relatively lower miR-30e* expression and that miR-182 directly suppressed cylindromatosis (CYLD), an NF-κB negative regulator. This suppression of CYLD promoted ubiquitin conjugation of NF-κB signaling pathway components and induction of an aggressive phenotype of glioma cells both in vitro and in vivo. Furthermore, we found that TGF-β induced miR-182 expression, leading to prolonged NF-κB activation. Importantly, the results of these experiments were consistent with an identified significant correlation between miR-182 levels with TGF-β hyperactivation and activated NF-κB in a cohort of human glioma specimens. These findings uncover a plausible mechanism for sustained NF-κB activation in malignant gliomas and may suggest a new target for clinical intervention in human cancer.     

TGF-β in the pathogenesis and prevention of disease: a matter of aneurysmic proportions

TGF-β regulates many aspects of cellular performance relevant to tissue morphogenesis and homeostasis. Postnatal perturbation of TGF-β signaling contributes to the pathogenesis of many disease states, as recently exemplified through the study of Marfan syndrome (MFS), including aortic aneurysm and skeletal muscle myopathy. Heterogeneity in the regulation and consequences of TGF-β signaling, amplified in the context of disease, has engendered confusion and controversy regarding its utility as a therapeutic target. Three studies recently published in the JCI, including one in this issue, underscore the complexity of this subject. Heydemann and colleagues implicate dimorphic variation in latent TGF-β–binding protein 4 (LTBP4), a regulator of TGF-β bioavailability and activation, as a modifier of muscular dystrophy in γ-sarcoglycan–deficient mice. In contrast to experience with ascending aortic aneurysm in MFS, Wang and colleagues show that systemic abrogation of TGF-β signaling worsens (rather than attenuates) Ang II–induced abdominal aortic aneurysm progression in mice. Tieu and colleagues define alterations in the regulation of vascular inflammation in the pathogenesis of Ang II–induced aneurysm and dissection in mice, which may help shed some light on this apparent paradox. Historically, perturbation (largely enhancement) of TGF-β signaling has been strongly implicated in the pathogenesis of diverse disease states, prominently including the initiation and progression of cancer and tissue fibrosis. Despite decades of intensive study in these contexts, the net effects of TGF-β signaling in disease pathogenesis and, perhaps more importantly, the incorporation of its antagonism into therapeutic strategies, remain controversial. This is nicely illustrated by the so-called TGF-β cancer paradox. In brief, TGF-β plays a prominent role in the suppression of tumorigenesis through induction of cell-cycle arrest and apoptosis and maintenance of cellular differentiation, as evidenced by the frequent biallelic loss of genes encoding TGF-β receptors or intracellular mediators of signaling in multiple tumor types (for example, see ref. 1). Attenuation or loss of tumor responsiveness can induce upregulation of TGF-β ligand expression, resulting in excessive stimulation of the signaling-intact neighboring stroma (reviewed in ref. 2). Attributable consequences include impairment of tumor surveillance through inhibition of adaptive immunity, acceleration of tumor growth through enhancement of angiogenesis, and induction of tumor invasion and metastasis through stimulation of innate immunity (including mast cell, monocyte, and macrophage chemotaxis), compromising of endothelial boundaries, and promotion of epithelial- or endothelial-mesenchymal transition (EpMT [ref. 2] and EnMT [ref. 3], respectively; collectively EMT). Amplification of TGF-β signaling can occur due to enhanced TGF-β ligand expression (e.g., by recruited mast cells or macrophages) or activation (e.g., by MMPs or simple fibrosis-dependent enhancement of shear stress within the tumor microenvironment; ref. 4). Additional TGF-β–related paradoxes are evident. For example, TGF-β can either induce or suppress angiogenesis depending upon its concentration and repertoire of TGF-β receptors and accessory proteins (2).     

Cytokine therapy reverses NK cell anergy in MHC-deficient tumors

Various cytokines have been evaluated as potential anticancer drugs; however, most cytokine trials have shown relatively low efficacy. Here, we found that treatments with IL-12 and IL-18 or with a mutant form of IL-2 (the “superkine” called H9) provided substantial therapeutic benefit for mice specifically bearing MHC class I–deficient tumors, but these treatments were ineffective for mice with matched MHC class I⁺ tumors. Cytokine efficacy was linked to the reversal of the anergic state of NK cells that specifically occurred in MHC class I–deficient tumors, but not MHC class I⁺ tumors. NK cell anergy was accompanied by impaired early signal transduction and was locally imparted by the presence of MHC class I–deficient tumor cells, even when such cells were a minor population in a tumor mixture. These results demonstrate that MHC class I–deficient tumor cells can escape from the immune response by functionally inactivating NK cells, and suggest cytokine-based immunotherapy as a potential strategy for MHC class I–deficient tumors. These results suggest that such cytokine therapies would be optimized by stratification of patients. Moreover, our results suggest that such treatments may be highly beneficial in the context of therapies to enhance NK cell functions in cancer patients.     

Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against β3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti–β3 integrin–mediated, but not anti-GPIbα–mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti–β3 integrin–mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti–β3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti–β3 integrin antisera and human anti–HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.