Activation of a dendritic cell-T cell axis by Ad5 immune complexes creates an improved environment for replication of HIV in T cells.

The STEP HIV vaccine trial, which evaluated a replication-defective adenovirus type 5 (Ad5) vector vaccine, was recently stopped. The reasons for this included lack of efficacy of the vaccine and a twofold increase in the incidence of HIV acquisition among vaccinated recipients with increased Ad5-neutralizing antibody titers compared with placebo recipients. To model the events that might be occurring in vivo, the effect on dendritic cells (DCs) of Ad5 vector alone or treated with neutralizing antiserum (Ad5 immune complexes [IC]) was compared. Ad5 IC induced more notable DC maturation, as indicated by increased CD86 expression, decreased endocytosis, and production of tumor necrosis factor and type I interferons. We found that DC stimulation by Ad5 IC was mediated by the Fcgamma receptor IIa and Toll-like receptor 9 interactions. DCs treated with Ad5 IC also induced significantly higher stimulation of Ad5-specific CD8 T cells equipped with cytolytic machinery. In contrast to Ad5 vectors alone, Ad5 IC caused significantly enhanced HIV infection in DC-T cell cocultures. The present results indicate that Ad5 IC activates a DC-T cell axis that, together with the possible persistence of the Ad5 vaccine in seropositive individuals, may set up a permissive environment for HIV-1 infection, which could account for the increased acquisition of HIV-1 infection among Ad5 seropositive vaccine recipients.

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.

Control of the adaptive response of the heart to stress via the Notch1 receptor pathway.

In the damaged heart, cardiac adaptation relies primarily on cardiomyocyte hypertrophy. The recent discovery of cardiac stem cells in the postnatal heart, however, suggests that these cells could participate in the response to stress via their capacity to regenerate cardiac tissues. Using models of cardiac hypertrophy and failure, we demonstrate that components of the Notch pathway are up-regulated in the hypertrophic heart. The Notch pathway is an evolutionarily conserved cell-to-cell communication system, which is crucial in many developmental processes. Notch also plays key roles in the regenerative capacity of self-renewing organs. In the heart, Notch1 signaling takes place in cardiomyocytes and in mesenchymal cardiac precursors and is activated secondary to stimulated Jagged1 expression on the surface of cardiomyocytes. Using mice lacking Notch1 expression specifically in the heart, we show that the Notch1 pathway controls pathophysiological cardiac remodeling. In the absence of Notch1, cardiac hypertrophy is exacerbated, fibrosis develops, function is altered, and the mortality rate increases. Therefore, in cardiomyocytes, Notch controls maturation, limits the extent of the hypertrophic response, and may thereby contribute to cell survival. In cardiac precursors, Notch prevents cardiogenic differentiation, favors proliferation, and may facilitate the expansion of a transient amplifying cell compartment.

Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor alpha gene.

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2R alpha gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4- CD8- cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4- CD8- thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

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.

H-2D ligand expression by Ly49A+ natural killer (NK) cells precludes ligand uptake from environmental cells: implications for NK cell function.

To study the adaptation of natural killer (NK) cells to their major histocompatibility complex (MHC) class I environment we have established a novel mouse model with mosaic expression of H-2D(d) using a Cre/loxP system. In these mice, we noticed that NK cells expressing the inhibitory receptor for D(d), Ly49A, were specifically underrepresented among cells with low D(d) levels. That was due to the acquisition of D(d) molecules by the Ly49A+ NK cells that have lost their D(d) transgene. The uptake of H-2D molecules via the Ly49A receptor was restricted to strong ligands of Ly49A. Surprisingly, when Ly49A+ NK cells were D(d+), uptake of the alternative ligand D(k) was not detectable. Similarly, one anti-Ly49A mAb (A1) bound inefficiently when Ly49A was expressed on D(d+) NK cells. Concomitantly, functional assays demonstrated a reduced capacity of Ly49A to inhibit H-2(b)D(d) as compared with H-2(b) NK cells, rendering Ly49A+ NK cells in D(d+) mice particularly reactive. Minor reductions of D(d) levels and/or increases of activating ligands on environmental cells may thus suffice to abrogate Ly49A-mediated NK cell inhibition. The mechanistic explanation for all these phenomena is likely the partial masking of Ly49A by D(d) on the same cell via a lateral binding site in the H-2D(d) molecule.

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

The Nlrp3 inflammasome regulates acute graft-versus-host disease.

The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome-mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro-IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication.

Attenuation of colon inflammation through activators of the retinoid X receptor (RXR)/peroxisome proliferator-activated receptor gamma (PPARgamma) heterodimer. A basis for new therapeutic strategies.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARgamma and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARgamma(1/)- and RXRalpha(1/)- mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARgamma heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARgamma agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARgamma and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor alpha and interleukin 1beta mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor kappaB DNA binding activity, c-Jun NH(2)-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARgamma and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARgamma heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARgamma ligands might hold promise in the clinic due to their synergistic effects.

T cell receptor specificity is critical for the development of epidermal gammadelta T cells.

A particular feature of gammadelta T cell biology is that cells expressing T cell receptor (TCR) using specific Vgamma/Vdelta segments are localized in distinct epithelial sites, e.g., in mouse epidermis nearly all gammadelta T cells express Vgamma3/Vdelta1. These cells, referred to as dendritic epidermal T cells (DETC) originate from fetal Vgamma3+ thymocytes. The role of gammadelta TCR specificity in DETC's migration/localization to the skin has remained controversial. To address this issue we have generated transgenic (Tg) mice expressing a TCR delta chain (Vdelta6.3-Ddelta1-Ddelta2-Jdelta1-Cdelta), which can pair with Vgamma3 in fetal thymocytes but is not normally expressed by DETC. In wild-type (wt) Vdelta6.3Tg mice DETC were present and virtually all of them express Vdelta6.3. However, DETC were absent in TCR-delta(-/-) Vdelta6.3Tg mice, despite the fact that Vdelta6.3Tg gammadelta T cells were present in normal numbers in other lymphoid and nonlymphoid tissues. In wt Vdelta6.3Tg mice, a high proportion of in-frame Vdelta1 transcripts were found in DETC, suggesting that the expression of an endogenous TCR-delta (most probably Vdelta1) was required for the development of Vdelta6.3+ epidermal gammadelta T cells. Collectively our data demonstrate that TCR specificity is essential for the development of gammadelta T cells in the epidermis. Moreover, they show that the TCR-delta locus is not allelically excluded.

Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen (SAg) that predominantly interacts with V(beta)8+ T cells. In vivo treatment of mice with SEB leads to an initial increase in the percentage of V(beta)8+ T cells, followed by a decrease in the numbers of these cells, eventually reaching lower levels than those found before treatment with the SAg. This decrease is due to apoptosis of the SEB-responding cells. In the present study, we use the distinct light scattering characteristics of apoptotic cells to characterize T cells that are being deleted in response to SEB in vivo. We show that dying, SEB-reactive T cells express high levels of Fas and Fas ligand (Fas-L), which are implicated in apoptotic cell death. In addition, the B cell marker B220 is upregulated on apoptotic cells. Moreover, we show that the generation of cells with an apoptotic phenotype is severely impaired in response to SEB in functional Fas-L-deficient mutant gld mice, confirming the role of the Fas pathway in SAg mediated peripheral deletion in vivo.

Granzyme A is an interleukin 1 beta-converting enzyme.

Apoptosis is critically dependent on the presence of the ced-3 gene in Caenorhabditis elegans, which encodes a protein homologous to the mammalian interleukin (IL)-1 beta-converting enzyme (ICE). Overexpression of ICE or ced-3 promotes apoptosis. Cytotoxic T lymphocyte-mediated rapid apoptosis is induced by the proteases granzyme A and B. ICE and granzyme B share the rare substrate site of aspartic acid, after which amino acid cleavage of precursor IL-1 beta (pIL-1 beta) occurs. Here we show that granzyme A, but not granzyme B, converts pIL-1 beta to its 17-kD mature form. Major cleavage occurs at Arg120, four amino acids downstream of the authentic processing site, Asp116. IL-1 beta generated by granzyme A is biologically active. When pIL-1 beta processing is monitored in lipopolysaccharide-activated macrophage target cells attacked by cytotoxic T lymphocytes, intracellular conversion precedes lysis. Prior granzyme inactivation blocks this processing. We conclude that the apoptosis-inducing granzyme A and ICE share at least one downstream target substrate, i.e., pIL-1 beta. This suggests that lymphocytes, by means of their own converting enzyme, could initiate a local inflammatory response independent of the presence of ICE.

Hierarchy of Notch-Delta interactions promoting T cell lineage commitment and maturation.

Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1-DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.