Wiskott–Aldrich syndrome protein deficiency in natural killer and dendritic cells affects antitumor immunity

Wiskott–Aldrich syndrome (WAS) is a primary immunodeficiency caused by reduced or absent expression of the WAS protein (WASP). WAS patients are affected by microthrombocytopenia, recurrent infections, eczema, autoimmune diseases, and malignancies. Although immune deficiency has been proposed to play a role in tumor pathogenesis, there is little evidence on the correlation between immune cell defects and tumor susceptibility. Taking advantage of a tumor‐prone model, we show that the lack of WASP induces early tumor onset because of defective immune surveillance. Consistently, the B16 melanoma model shows that tumor growth and the number of lung metastases are increased in the absence of WASP. We then investigated the in vivo contribution of Was^?/? NK cells and DCs in controlling B16 melanoma development. We found fewer B16 metastases developed in the lungs of Was^?/? mice that had received WT NK cells as compared with mice bearing Was^?/? NK cells. Furthermore, we demonstrated that Was^?/? DCs were less efficient in inducing NK‐cell activation in vitro and in vivo. In summary, for the first time, we demonstrate in in vivo models that WASP deficiency affects resistance to tumor and causes impairment in the antitumor capacity of NK cells and DCs.     

Defective actin polymerization in EBV-transformed B-cell lines from patients with the Wiskott–Aldrich syndrome

The Wiskott–Aldrich syndrome (WAS) is a rare X-linked recessive disorder characterized by eczema, thrombocytopenia, and immunodeficiency. An allelic variant of the disease is characterized by isolated thrombocytopenia (XLT). The gene responsible for WAS/XLT (WASP) encodes for a 502 amino acid protein (WASP) that is possibly involved in actin binding and cytoskeleton organization. The expression of WASP and the distribution of F-actin and alpha-actinin (which binds to and stabilizes actin filaments) have been analysed in lymphoblastoid cell lines from six patients with WAS and one with XLT. Western blot and immunocytochemistry did not reveal WASP expression in four WAS patients, whereas two WAS patients (with a moderate clinical course) expressed trace amounts of mutant WASP. In contrast, the XLT patient expressed normal amounts of WASP. Furthermore, cell lines from WAS and XLT patients also markedly differed in F-actin polymerization and alpha-actinin distribution. In particular, severe defects of cytoplasmic F-actin expression and of F-actin-positive microvillus formation, and impaired capping of alpha-actinin, were observed in all patients who lacked WASP. As a whole, the degree of impairment of WASP protein expression in WAS/XLT seems to correlate with anomalies of cytoskeletal organization, strongly supporting a role for WASP in the regulation of F-actin polymerization. © 1998 John Wiley & Sons, Ltd.     

DC therapy induces long‐term NK reactivity to tumors via host DC

We vaccinated mice with DC loaded with or without invariant NKT‐cell ligand α‐galactosylceramide and evaluated long‐term resistance against tumor challenge. When mice had been given either DC or DC/galactosylceramide and were challenged with tumor cells even 6–12 months later, both NK and NKT cells were quickly activated to express CD69 and produce IFN‐γ. The NK cells could resist a challenge with several different tumors in vivo. The activated NK and NKT cells could be depleted with anti‐NK1.1 treatment. In spite of this, the activated cells recovered, indicating that tumor‐responsive NK and NKT cells were being generated continuously as a result of vaccination with DC and were not true memory cells. The NK and NKT antitumor response in DC‐vaccinated mice depended on CD4⁺ T cells, but neither CD8⁺T cells nor CD4⁺CD25⁺ regulatory T cells. However, both vaccine DC and host DC were required for the development of long‐term, tumor reactive innate immunity. These results indicate that DC therapy in mice induces long‐lasting innate NK‐ and NKT‐cell activation through a pathway that requires host DC and CD4⁺ T cells and that the continued generation of active NK cells resists the establishment of metastases in vivo.     

Exacerbated experimental arthritis in Wiskott–Aldrich syndrome protein deficiency: Modulatory role of regulatory B cells

Patients deficient in the cytoskeletal regulator Wiskott–Aldrich syndrome protein (WASp) are predisposed to varied autoimmunity, suggesting it has an important controlling role in participating cells. IL‐10‐producing regulatory B (Breg) cells are emerging as important mediators of immunosuppressive activity. In experimental, antigen‐induced arthritis WASp‐deficient (WASp knockout [WAS KO]) mice developed exacerbated disease associated with decreased Breg cells and regulatory T (Treg) cells, but increased Th17 cells in knee‐draining LNs. Arthritic WAS KO mice showed increased serum levels of B‐cell‐activating factor, while their B cells were unresponsive in terms of B‐cell‐activating factor induced survival and IL‐10 production. Adoptive transfer of WT Breg cells ameliorated arthritis in WAS KO recipients and restored a normal balance of Treg and Th17 cells. Mice with B‐cell‐restricted WASp deficiency, however, did not develop exacerbated arthritis, despite exhibiting reduced Breg‐ and Treg‐cell numbers during active disease, and Th17 cells were not increased over equivalent WT levels. These findings support a contributory role for defective Breg cells in the development of WAS‐related autoimmunity, but demonstrate that functional competence in other regulatory populations can be compensatory. A properly regulated cytoskeleton is therefore important for normal Breg‐cell activity and complementation of defects in this lineage is likely to have important therapeutic benefits.     

B‐cell intrinsic TLR7 signals promote depletion of the marginal zone in a murine model of Wiskott–Aldrich syndrome

Patients with Wiskott–Aldrich syndrome (WAS) exhibit prominent defects in splenic marginal zone (MZ), resulting in abnormal T‐cell‐independent antibody responses and increased bacterial infections. B‐cell‐intrinsic deletion of the affected gene WAS protein (WASp) markedly reduces splenic MZ B cells, without impacting the rate of MZ B‐cell development, suggesting that abnormal B‐cell retention within the MZ accounts for MZ defects in WAS. Since WASp regulates integrin‐dependent actin cytoskeletal rearrangement, we previously hypothesized that defective B‐cell integrin function promotes MZ depletion. In contrast, we now report that B‐cell‐intrinsic deletion of the TLR signaling adaptor MyD88 is sufficient to restore the MZ in WAS. We further identify TLR7, an endosomal single‐stranded RNA (ssRNA) receptor, as the MyD88‐dependent receptor responsible for WAS MZ depletion. These findings implicate spontaneous activation of MZ B cells by ssRNA‐containing self‐ligands (likely derived from circulating apoptotic material) as the mechanism underlying MZ depletion in WAS. Together, these data suggest a previously unappreciated role for B‐cell intrinsic TLR signals in MZ homeostasis, of relevance to both pathogen responses and to the development of systemic autoimmunity.     

Site‐specific anti‐tumor immunity: Differences in DC function,TGF‐β production and numbers of intratumoral Foxp3+ Treg

Gliomas localized within the CNS are generally not rejected by the immune system despite being immunogenic. This failure of the immune system has been associated both with glioma‐derived immunosuppressive molecules and the immune‐privileged state of the CNS. However, the relative contribution of tumor location to the glioma‐mediated immunosuppression, as well as the immune mechanisms involved in the failure of glioma rejection are not fully defined. We report here that syngeneic GL261 gliomas growing either intracranially or subcutaneously in mice are infiltrated by DC and T cells. However, only subcutaneous gliomas elicit an effective anti‐tumor immune response. In contrast to DC infiltrating subcutaneously grown GL261 gliomas, tumor‐infiltrating DC from intracranial gliomas do not activate antigen‐dependent T‐cell proliferation in vitro. In addition, brain‐localized GL261 gliomas are characterized by significantly higher numbers of Foxp3⁺ Treg and higher levels of TGF‐β1 mRNA and protein expression when compared with GL261 gliomas in the skin. Our data show that gliomas in the CNS, but not in the skin, give rise to TGF‐β production and accumulation of both Treg and functionally impaired DC. Thus, not the tumor itself, but its location dictates the efficiency of the anti‐tumor immune response.     

Klebsiella pneumoniae‐triggered DC recruit human NK cells in a CCR5‐dependent manner leading to increased CCL19‐responsiveness and activation of NK cells

Besides their role in destruction of altered self‐cells, NK cells have been shown to potentiate T‐cell responses by interacting with DC. To take advantage of NK–DC crosstalk in therapeutic DC‐based vaccination for infectious diseases and cancer, it is essential to understand the biology of this crosstalk. We aimed to elucidate the in vitro mechanisms responsible for NK‐cell recruitment and activation by DC during infection. To mimic bacterial infection, DC were exposed to a membrane fraction of Klebsiella pneumoniae, which triggers TLR2/4. DC matured with these bacterial fragments can actively recruit NK cells in a CCR5‐dependent manner. An additional mechanism of DC‐induced NK‐cell recruitment is characterized by the induction of CCR7 expression on CD56^dimCD16⁺ NK cells after physical contact with membrane fraction of K. pneumoniae‐matured DC, resulting in an enhanced migratory responsiveness to the lymph node‐associated chemokine CCL19. Bacterial fragment‐matured DC do not only mediate NK‐cell migration but also meet the prerequisites needed for augmentation of NK‐cell cytotoxicity and IFN‐γ production, the latter of which contributes to Th1 polarization.     

DNAX accessory molecule‐1 (CD226) promotes human hepatocellular carcinoma cell lysis by Vγ9Vδ2 T cells

Human Vγ9Vδ2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway‐derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate. They display a strong cytotoxic activity against several tumor types, including hepatocellular carcinoma (HCC). Little is known about the mechanisms underlying Vγ9Vδ2 T‐cell recognition of tumor cells, but there is strong evidence that activating NK receptors play a role in γδ T‐cell cytotoxicity. In this study, we showed that the two NK receptors DNAX accessory molecule‐1 (DNAM‐1) and CD96 were expressed by Vγ9Vδ2 T cells. The ligands Nectin‐like‐5 specific of both DNAM‐1 and CD96, and also Nectin‐2, an additional ligand of DNAM‐1, were present on all HCC cell lines analyzed. Furthermore, we demonstrated by mAb‐mediated masking experiments that cytotoxicity against HCC cells as well as IFN‐γ production in γδ T cells were dependent on DNAM‐1. Our experiments indicated that Nectin‐like‐5 but not Nectin‐2 was involved in DNAM‐1‐dependent γδ T‐cell functions. We did not reveal a role for CD96 in the killing of HCC cells. Finally, we showed by combined mAb‐mediated blockade that DNAM‐1 and NKG2D could cooperate in the cell lysis of HCC.     

Wiskott–Aldrich Syndrome: Immunodeficiency resulting from defective cell migration and impaired immunostimulatory activation

Regulation of the actin cytoskeleton is crucial for many aspects of correct and cooperative functioning of immune cells, such as migration, antigen uptake and cell activation. The Wiskott–Aldrich Syndrome protein (WASp) is an important regulator of actin cytoskeletal rearrangements and lack of this protein results in impaired immune function. This review discusses recent new insights of the role of WASp at molecular and cellular level and evaluates how WASp deficiency affects important immunological features and how defective immune cell function contributes to compromised host defence.     

IFN‐λ‐mediated IL‐12 production in macrophages induces IFN‐γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.     

Innate IFN‐γ promotes development of experimental autoimmune encephalomyelitis: A role for NK cells and M1 macrophages

The role of IFN‐γ in the pathogenesis of autoimmune diseases is controversial. Although Th1 cells can induce experimental autoimmune encephalomyelitis (EAE), IFN‐γ can suppress Th17 cells that are pathogenic in EAE. Here we show that NK cells provide an early source of IFN‐γ during development of EAE. Depletion of NK cells or neutralization of IFN‐γ delayed the onset of EAE and was associated with reduced infiltration of IL‐17⁺ and GM‐CSF⁺ T cells into the CNS. In the passive transfer model, immune cells from myelin oligodendrocyte glycoprotein (MOG)‐immunized IFN‐γ^?/? mice failed to induce EAE, despite producing IL‐17 and GM‐CSF. The macrophages expressed markers of M2 activation and the T cells had low very late antigen‐4 (VLA‐4) expression and failed to infiltrate the CNS. Addition of recombinant IFN‐γ to immune cells from the IFN‐γ^?/? mice activated M1 macrophages and restored VLA‐4 expression, migratory, and encephalitogenic activity of T cells. Furthermore, treatment of recipient mice with anti‐VLA‐4 neutralizing antibody abrogated EAE induced by transfer of T cells from WT mice. Our findings demonstrate IFN‐γ‐producing T cells are not required for development of EAE, but NK cell‐derived IFN‐γ has a key role in promoting M1 macrophage expansion and VLA‐4‐mediated migration of encephalitogenic T cells into the CNS.     

Microfluidic‐based,live‐cell analysis allows assessment of NK‐cell migration in response to crosstalk with dendritic cells

Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell–DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo‐repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single‐cell level, C57BL/6 NK‐cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS‐activated mature DCs induced a high level of chemotactic movement of IL‐2‐activated NK cells in vitro. We confirmed these findings in a standard trans‐well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony‐stimulating factor in repulsing NK‐cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK‐cell migratory responses in NK cell–DC crosstalk will provide new insights into the development of DC‐based NK‐cell therapies against tumor and infections.     

CCL21‐expression and accumulation of CCR7+ NK cells in livers of patients with primary sclerosing cholangitis

The pathogenesis of primary sclerosing cholangitis (PSC), an autoimmune liver disease, remains unknown. The aim of this study was to characterize peripheral blood and intrahepatic NK cells from patients with PSC. Peripheral blood samples from patients with PSC, other autoimmune liver diseases, and from healthy control individuals were used, as well as liver tissues from PSC patients undergoing liver transplantation. Multiparameter flow cytometry showed that peripheral blood NK cells from PSC patients were significantly enriched for CCR7⁺ and CXCR3⁺ cells, and CCR7⁺ but not CXCR3⁺ cells were also significantly increased within intrahepatic NK cells. PSC patients undergoing liver transplantation furthermore had significantly higher plasma levels of the CCR7‐ligand CCL21, and the CXCR3‐ligands CXCL10 and CXCL11, and significantly higher levels of CCL21, but not CXCL10, were detected in liver tissues. CCR7⁺ and CXCR3⁺ NK cells from PSC patients exhibited significantly higher functional capacity in peripheral blood, but not liver tissues, consistent with chronic activation of these NK cells in the inflamed liver. These data show that PSC is characterized by intrahepatic CCL21 expression and accumulation of CCR7⁺ NK cells in the inflamed liver tissue.     

H2‐M3‐restricted CD8+ T cells augment CD4+ T‐cell responses by promoting DC maturation

Infection with Listeria monocytogenes triggers the activation and expansion of nonconventional CD8⁺ T cells restricted by the MHC class Ib molecule, H2‐M3. H2‐M3‐restricted CD8⁺ T cells exhibit a memory phenotype, rapidly produce cytokines, and reach peak frequencies sooner than conventional MHC class Ia‐restricted CD8⁺ T cells. In this study, we found that simultaneous in vivo priming of H2‐M3‐restricted T cells and adoptively transferred OT‐II CD4⁺ T cells on the same DC enhances the survival of OT‐II cells. Stimulation of H2‐M3‐restricted T cells were found to induce DC maturation resulting in costimulatory molecule upregulation and production of T_H1‐type cytokines, which was dependent on both cell‐to‐cell contact and soluble factors, particularly TNF‐α, produced by activated H2‐M3‐restricted T cells. Interestingly, H2‐M3‐restricted T cells were more efficient than activated NK cells in inducing DC maturation. Furthermore, we found that OVA_323–339‐coated DC matured by coculturing with peptide‐stimulated H2‐M3‐restricted T cells were more efficient in stimulating the proliferation of Ag‐activated OT‐II cells. This study indicates that H2‐M3‐restricted T cells promote immune responses by CD4⁺ T cells by inducing DC maturation and suggests novel mechanisms for vaccine development.     

Cover Picture: Involvement of nonclassical MHC class Ib molecules in heat shock protein‐mediated anti‐tumor responses – Eur. J. Immunol. 6/2007

The image of the jumping frog was kindly provided by Jacques Robert and created by his son by Adrien. Anti‐tumor responses, both in vitro and in vivo, are studied using Xenopus laevis by Robert and colleagues (pp. 1494–1501); non‐classical MHC class Ib molecules are shown, by RNA interference, to be important for the gp96‐induced CTL response against class Ia‐negative tumors in Xenopus. The conservation of the class Ia‐unrestricted response from Xenopus to mammals indicates the importance of this anti‐tumor immune mechanism.     

IFN‐γ production by CD27+ NK cells exacerbates Listeria monocytogenes infection in mice by inhibiting granulocyte mobilization

Natural killer (NK) cells are key components of the immune system involved in several immune reactions, including the clearance of intracellular pathogens. When activated, NK cells rapidly secrete particular cytokines that activate innate immunity and facilitate development of adaptive responses. Conflicting reports on the role of NK cells during infection by Listeria monocytogenes can be found in the literature. Here, we demonstrate that during lethal infection by L. monocytogenes, activation of NK cells via the costimulatory molecule CD27 leads to excessive IFN‐γ production. This impairs innate anti‐bacterial host defenses by inducing downregulation of CXCR2 on granulocytes and consequently inhibiting their recruitment to the sites of infection. The use of antibodies to block CD27 signaling or to deplete IFN‐γ was sufficient to rescue mice from lethal challenge by L. monocytogenes. Our findings contribute to a better understanding of the importance of CD27 signaling in activation of NK cells and should provide new ways of interfering with infections.