Plasmacytoid dendritic cells: one-trick ponies or workhorses of the immune system?

Plasmacytoid dendritic cells (pDCs) were first described as interferon-producing cells and, for many years, their overlapping characteristics with both lymphocytes and classical dendritic cells (cDCs) created confusion over their exact ontogeny. In this Viewpoint article, Nature Reviews Immunology asks five leaders in the field to discuss their thoughts on the development and functions of pDCs--do these cells serve mainly as a major source of type I interferons or do they also make other important contributions to immune responses?

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Lack of directed V alpha 14-J alpha 281 rearrangements in NK1+ T cells.

NK1.1+ T cells are an unusual subset of TCR alpha beta cells distinguished by their highly restricted V beta repertoire and predominant usage of an invariant V alpha 14-J alpha 281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant alpha chain, we have analyzed V alpha 14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T-T hybrids derived from thymic NK1.1+ T cells. As expected a high proportion (17/20) of the hybrids had rearranged V alpha 14 to J alpha 281. However, V alpha 14-J alpha 281 rearrangements always occurred on only one chromosome and were accompanied by other V alpha-J alpha rearrangements (not involving V alpha 14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of V alpha 14-J alpha 281 rearrangements in NK1.1+ T cells.

Selective lysis of activated cells in oncorna virus infection.

The authors devised a cytotoxic assay based on cytofluorometric analysis of target surface markers in order to compare lysis exerted in vitro by cytotoxic T lymphocytes (CTLs) on different cell subsets in the context of a single lymphoid target cell population. Using this assay, the authors evaluated when oncorna virus-infected lymphocytes become a suitable target for virus-specific T cell effectors. A lymphocyte population from Moloney-murine leukaemia virus (M-MuLV)-infected (carrier) mice, in which the proliferation of selective V beta T-cell receptor (TCR) families was induced in response to Mlsa encoded antigens, was utilized as a target. The authors observed that a virus-specific T cell clone exerted lytic activity preferentially against activated cell subsets. Moreover, virus-specific CTLs generated in mixed leucocyte tumour cell cultures (MLTC) were also able to impair the concomitant anti-Mlsa response of lymphocytes from M-MuLV carrier mice. It was found that the proliferative status of oncorna virus-infected target cells played an important role in limiting the in vitro efficacy of the immune response, and it is speculated that this phenomenon might represent an in vivo escape mechanism from immunosurveillance.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Cutting edge: apoptosis of superantigen-activated T cells occurs preferentially after a discrete number of cell divisions in vivo.

Staphylococcal enterotoxins are bacterial products that display superantigen activity in vitro as well as in vivo. For instance, staphylococcal enterotoxin B (SEB) polyclonally activates T cells that bear the Vbeta8 gene segment of the TCR. SEB-activated T cells undergo a burst of proliferation that is followed by apoptosis. Using an in vivo adaptation of a fluorescent cell division monitoring technique, we show here that SEB-activated T cells divide asynchronously, and that apoptosis of superantigen-activated T cells is preferentially restricted to cells which have undergone a discrete number of cell divisions. Collectively, our data suggest that superantigen-activated T cells are programmed to undergo a fixed number of cell divisions before undergoing apoptosis. A delayed death program may provide a mechanistic compromise between effector functions and homeostasis of activated T cells.

Insights into the regulation of two caspase-activating platforms, the inflammasome and the PIDDosome

Résumé: Les organismes multicellulaires ont adopté diverses stratégies pour répondre aux stress auxquels ils sont exposés. Cette étude a exploré deux de ces stratégies l'inflammation en réponse à une invasion par un pathogène, et l'apoptose ou la survie en réponse aux dommages à l'ADN. L'interleukine-lß (IL-lß) est une importante cytokine inflammatoire. Elle est synthétisée sous forme d'un précurseur inactif et nécessite un clivage par la caspase-1 pour être activée. La caspase-1 elle-même est activée dans un complexe appelé inflammasome. Certains NLRs (Nod-like receptors), IPAF et les NALPs, sont capables de former des inflammasomes fonctionnels. Cette étude s'est intéressée au rôle d'un autre NLR structurellement proche, la protéine NAIP, dans la régulation de la caspase-1 et la maturation de l'IL-1 ß. NAIP est incorporé à l'inflammasome contenant NALP3 et est capable d'inhiber l'activation de la caspase-1 et la maturation de l'IL-lß. Cette fonction inhibitrice dépend des ses domaines BIR et est inhibée par ses LRRs. Le mécanisme exact d'inhibition reste à définir et la régulation de l'activation de NAIP est discutée. La deuxième partie de cette étude concerne la protéine PIDD. Cette protéine est impliquée avec RAIDD dans l'activation de la caspase-2, et est aussi capable, avec l'aide de RIP et de NEMO, d'activer NF-κB en réponse aux dommages à l'ADN. Deux isoformes de PIDD ont déjà été décrites dans la littérature, PIDD (isoforme 1) et LRDD (isoforme 2) et une troisième isoforme est rapportée ici. L'étude de l'expression de ces isoformes a montré qu'elles sont exprimées différemment dans les tissus et dans les lignées cellulaires, et que l'isoforme 3 est induite en réponse à un stress génotoxique. La caractérisation fonctionnelle a établi que les trois isoformes sont capables d'activer NF-κB, donc la survie, mais que seule l'isoforme 1 peut interagir avec RAIDD pour activer la caspase-2 et sensibiliser les cellules à la mort induite par un stress génotoxique. Le domaine intermédiaire de PIDD, situé entre le deuxième ZU5 et le DD est essentiel pour l'interaction entre PIDD et RAIDD et l'activation de la caspase-2 qui en découle. En conclusion, l'épissage différentiel de l'ARNm de PIDD permet la production d'au moins trois protéines possédant des fonctions agonistes ou antagonistes et qui peuvent participer au choix cellulaire entre survie et apoptose en réponse aux dommages à l'ADN. Summary: Multicellular organisms have evolved several strategies to cope with the stresses they encounter. The present study has explored two of these strategies: inflammation in response to a pathogenic invasion, and apoptosis or repair/survival in response to DNA damage. Interleukin-lß (IL-lß) is a key mediator of inflammation. It is synthesized as an inactive precursor and requires cleavage by caspase-1 to be activated. caspase-1 itself is activated in molecular platforms called inflammasomes, which can be formed by members of the NOD-like receptors (NLR) family, like IPAF and NALPs. This study has investigated the role of another NLR, the structurally related protein NAIP, in the regulation of caspase-1 activation and IL-lß maturation. An inhibitory role of NAIP on caspase-1 activation and IL-lß maturation was demonstrated, as well as NAIP incorporation in the NALP3 inflammasome. This inhibitory property relies on NAIP BIR domains and is inhibited by NAIP LRRs. The exact mechanism of NAIP-mediated caspase-1 activation remains to be elucidated and the regulation of NAIP activation is discussed. The second part of this study focused on the caspase-2 activating protein PIDD. This protein is known to mediate caspase-2 activation via RAIDD and to signal NF-κB via RIP and NEMO in response to DNA damage. Two isoforms of PIDD, PIDD (isoform 1) and LRDD (isoform 2), have already been reported and a third isoform is described here. Investigation of the expressional regulation of these isoforms indicated that they are differentially expressed in tissues and cell lines, and that isoform 3 mRNA levels are upregulated in response to genotoxic stress. Functional studies demonstrated that all three isoforms can activate NF-κB in response to DNA damage, but only isoform 1 is able to interact with RAIDD and activate caspase-2, sensitizing cells to genotoxic stress-induced cell death. The intermediate domain located between the second ZUS and the DD is essential for the interaction of PIDD and RAIDD and the subsequent caspase-2 activation. Thus the differential splicing of PIDD mRNA leads to the formation of at least thrée proteins with antagonizing/agonizing functions that could participate in determining cell fate in response to DNA damage.

The role of the Estrogen-Responsive B box protein in skin morphogenesis, wound repair and skin disease

SUMMARY: EBBP is a poorly characterized member of the RBCC/TRIM family (RING finger B-box coiled-coilltripartite motif). It is ubiquitously expressed, but particularly high levels are found in keratinocytes. There is evidence that EBBP is involved in inflammatory processes, since it can interact with pro-interleukin-1 ß (prolL-1 ß) in human macrophages and keratinocytes, and its downregulation results in reduced secretion of IL-1 ß. IL-1ß activation and secretion requires the proteolytic cleavage of prolL-1ß by caspase-1, which in turn is actìvated by a protein complex called the inflammasome. As it has been demonstrated that EBBP can bind two different proteins of the inflammasome (NALP-1 and caspase 1), we assumed that EBBP plays a role in the regulation of inflammation and that the inflammasome, which has as yet only been described in ínflammatory cells, may also exist in keratinocytes. Indeed, I could show in my thesis that the inflammasome components are expressed in human keratinócytes at the RNA and protein level and also in vivo in human epidermis. After irradiation with a physiological dose of UVB, keratinocytes activated prolL-1ß and secreted prolL-1 a, IL-1 ß, prolL-18 and inflammasome proteins, although all these proteins lack a classical signal peptide. The secretion was dependent on caspase-1 activity, but not on de novo protein synthesis. Knock-down of NALP1 and -3, caspase-1 and -5, EBBP and Asc strongly reduced the secretion of IL-1 ß, demonstrating that also in keratinocytes inflammasome proteins are directly involved in maturation of this cytokine. These results demonstrate for the first time the presence of an active inflammasome in non-professional immune cells. Moreover, they show that UV irradiation is a stimulus for inflammasome activation in keratinocytes. For the analysis of the ín vivo functions of EBBP, transgenic mice overexpressing EBBP in the epidermis were generated. To examine the influence of EBBP overexpression on inflammatory processes, we subjected the mice to different challenges, which induce inflammation. Wound-healing, UVB irradiation and delayed hypersensitivity were tested, but we did not observe any phenotype in the K14-EBBP mice. Besides, a conditional ebbp knockout mouse has been obtained, which will allow to determine the effects of EBBP gene deletion in different tissues and organs. RESUME: EBBP est un membre encore mal connu de la famille des RBCC/TRIM (RING finger B-box coiled-coil/tripartite motif). Il est exprimé de manière ubiquitaire, et en particulier dans les kératinocytes. EBBP étant capable d'interagir avec la prointerleukine-1 ß (prolL-1 ß) dans les macrophages et les kératinocytes humains et de réguler la sécrétion de l'IL-1 ß, il est très probable que cette protéine est impliquée dans l'inflammation. L'activation et la sécrétion de l'IL-1 ß requièrent le clivage protéolytique de son précurseur prolL-1ß par la caspase-1, qui est elle-même activée par un complexe protéique appelé l'inflammasome. Comme il a été démontré qu'EBBP peut lier deux protéines de l'inflammasome (NALP-1 et caspase-1), nous avons émis l'hypothèse qu'EBBP joue un rôle dans la régulation de l'inflammation et que l'inflammasome, jusqu'ici décrit exclusivement dans des cellules inflammatoires, existe dans les kératinocytes. En effet, j'ai pu montrer dans ma thèse que les composants de l'inflammasome sont exprimés dans les kératinocytes humains ainsi que in vivo dans l'épiderme humain. Après irradiation avec une dose, physiologique d'UVB, les kératinocytes activent la prolL-1 ß et sécrètent la prolL-1a, l'IL-1 ß, la prolL-18 et des protéines de l'inflammasome, bien que toutes ces protéines soient dépourvues de peptide signal. La sécrétion dépend de la caspase-1 mais pas de la synthèse protéique de novo. Le knock-down de NALP-1 et -3, des caspase-1 et -5, d'EBBP et d'Asc réduit de manière marquée la sécrétion d'IL-1 ß, démontrant que dans les kératinocytes également, les protéines de l'inflammasome sont impliquées directement dans la maturation de cette cytokine. Ces résultats démontrent pour la première fois la présence d'un inflammasome actif dans des cellules immunitaires non professionnelles. De plus, ils montrent que l'irradiation aux UV est un stimulus pour l'activation de l'inflammasome dans les kératinocytes. Pour l'analyse des fonctions d'EBBP in vivo, nous avons généré des souris transgéniques qui surexpriment EBBP dans l'épiderme. En vue d'examiner l'influence de la surexpression d'EBBP sur le processus inflammatoire, nous avons soumis ces souris à differents modèles d'inflammation. Nous avons testé cicatrisation, UVB et hypersensibilité retardée, mais n'avons pas observé de phénotype chez les souris transgéniques. En parallèle, nous avons également généré des souris knock-out pour ebbp qui devraient nous permettre de déterminer les effets de la suppression d'EBBP dans différents tissus et organes.

Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells.

Interleukin 7 is essential for the survival of naive T lymphocytes. Despite its importance, its cellular source in the periphery remains poorly defined. Here we report a critical function for lymph node access in T cell homeostasis and identify T zone fibroblastic reticular cells in these organs as the main source of interleukin 7. In vitro, T zone fibroblastic reticular cells were able to prevent the death of naive T lymphocytes but not of B lymphocytes by secreting interleukin 7 and the CCR7 ligand CCL19. Using gene-targeted mice, we demonstrate a nonredundant function for CCL19 in T cell homeostasis. Our data suggest that lymph nodes and T zone fibroblastic reticular cells have a key function in naive CD4(+) and CD8(+) T cell homeostasis by providing a limited reservoir of survival factors.

Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes.

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.

Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells.

NK T cells produce cytokines when their semi-invariant TCR engages glycolipids associated with CD1d. The physiological consequences of NKT cell activation remain controversial, although they have been implicated in control of autoimmunity, parasites and tumors. We show here that specific activation of NKT cells in liver and spleen leads to a rapid induction of extensive NK cell proliferation and cytotoxicity. This NK cell activation is dependent, at least in part, on IFN-gamma production by NKT cells and IL-12 production by antigen-presenting cells. Remarkably, activation of NK cells by NKT cells is highly selective, since bystander T and B lymphocytes show transient expression of activation markers but almost no proliferation. Collectively our data suggest that CD1d-dependent NKT cells regulate innate immunity by sampling blood-borne glycolipid antigens and rapidly activating NK cells.

Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Transcriptional activation of the utrophin promoter B by a constitutively active Ets-transcription factor.

Duchenne muscular dystrophy is an X-linked genetic disease caused by the absence of functional dystrophin. Pharmacological upregulation of utrophin, the autosomal homologue of dystrophin, offers a potential therapeutic approach to treat Duchenne patients. Full-length utrophin mRNA is transcribed from two alternative promoters, called A and B. In contrast to the utrophin promoter A, little is known about the factors regulating the activity of the utrophin promoter B. Computer analysis of this second promoter revealed the presence of several conserved binding motives for Ets-transcription factors. Using electrotransfer of cDNA into mouse muscles, we demonstrate that a genetically modified beta-subunit of the Ets-transcription factor GA-binding protein potently activates a utrophin promoter B reporter construct in innervated muscle fibers in vivo. These results make the GA-binding protein and the signaling cascade regulating its activity in muscle cells, potential targets for the pharmacological modulation of utrophin expression in Duchenne patients.

Human urothelial cells grown on collagen adsorbed to surface-modified polymers.

OBJECTIVES: Tissue engineering methods can be applied to regenerate diseased, or congenitally missing, urinary tract tissues. Urinary tract tissue cell cultures must be established in vitro and adequate matrices, acting as cell carriers, must be developed. Although degradable and nondegradable polymer matrices offer adequate mechanical stability, they are not optimal for cell adherence and growth. To overcome this problem, extracellular matrix proteins, permitting cell adhesion and regulation of cell proliferation and differentiation, can be adsorbed to the surface-modified polymer. METHODS: In this study, nondegradable polymer films, poly(ethylene terephthalate), were used as an experimental model. Films were modified by graft polymerization of acrylic acid to subsequently allow collagen type I and III immobilization. The following adhesion, proliferation of human urothelial cells, and induction of their stratification were analyzed. RESULTS: Collagen adsorption on 0.2 microg/cm2 poly(acrylic acid)-grafted polymer films rendered the matrix apt for human urothelial cell adhesion and proliferation. Furthermore, stratification of urothelial cells was demonstrated on these surface-modified matrices. CONCLUSIONS: These results have shown that surface-modified polymer matrices can be used to act as cell carriers for cultured human urothelial cells. Such a cell-matrix construct could be applied in reparative surgery of the urinary tract.

Time-Course In Vivo Trafficking Pattern and Effector Function of Donor-Specific Regulatory T Cells in Response to an Allograft.

Background: Experimental data have suggested that adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs), capable of controlling immune responses to specifi c auto- or alloantigens, could be used as a therapeutic strategy to promote specifi c tolerance in T-cell mediated diseases and in organ transplantation (Tx). However, before advocating the application of immunotherapy with Tregs in Tx, we need to improve our understanding of their in vivo homeostasis, traffi cking pattern and effector function in response to alloantigens. Methods : Donor-antigen specifi c murine Tregs were generated and characterized in vitro following our described protocols. Using an adoptive transfer and skin allotransplantation model, we have analyzed the in vivo expansion and homing of fl uorescent-labeled effector T cells (Teff) and Tregs, at different time-points after Tx, using fl ow-cytometry as well as fl uorescence microscopy techniques. Results: Tregs expressed CD62L, CCR7 and CD103 allowing their homing into lymphoid and non-lymphoid tissues (gut, skin) after intravenous injection. While hyporesponsive to TCR stimulation in vitro, transferred Tregs survived, migrated to secondary lymphoid organs and preferentially expanded within the allograft draining lymph nodes. Furthermore, Foxp3+ cells could be detected inside the allograft as early as day 3-5 after Tx. At a much later time-point (day 60 after Tx), graft-infi ltrating Foxp3+ cells were also detectable in tolerant recipients. When transferred alone, CD4+CD25- Teff cells expanded within secondary lymphoid organs and infi ltrated the allograft by day 3-5 after Tx. The co-transfer of Tregs limited the expansion of alloreactive Teff cells as well as their recruitment into the allograft. The promotion of graft survival observed in the presence of Tregs was in part mediated by the inhibition of the production of effector cytokines by CD4+CD25- T cells. Conclusion: Taken together, our results suggest that the suppression of allograft rejection and the induction of Tx tolerance are in part dependant on the alloantigendriven homing and expansion of Tregs. Thus, the appropriate localization of Tregs may be critical for their suppressive function in vivo.