Unique lamina propria stromal cells imprint the functional phenotype of mucosal dendritic cells.

Mucosal dendritic cells (DCs) in the intestine acquire the unique capacity to produce retinoic acid (RA), a vitamin A metabolite that induces gut tropism and regulates the functional differentiation of the T cells they prime. Here, we identified a stromal cell (SC) population in the intestinal lamina propria (LP), which is capable of inducing RA production in DCs in a RA- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent fashion. Unlike DCs, LP SCs constitutively expressed the enzymatic machinery to produce RA even in the absence of dietary vitamin A, but were not able to do so in germ-free mice implying regulation by microbiota. Interestingly, DCs promoted GM-CSF production by the SCs indicating a two-way cross-talk between both cell types. Furthermore, RA-producing LP SCs and intestinal DCs localized closely in vivo suggesting that the interactions between both cell types might have an important role in the functional education of migratory DCs and therefore in the regulation of immune responses toward oral and commensal antigens.

Long-term endurance exercise decreases antiangiogenic endostatin signalling in overweight men aged 50-60 years.

BACKGROUND: Endurance training may decrease the risk of coronary artery disease. It has been speculated that these effects may be due to an exercise-induced stimulation of angiogenesis. The underlying mechanisms are not yet clear. Therefore, using ELISA, we investigated the plasma level of vascular endothelial growth factor (VEGF, angiogenic factor) and endostatin (antiangiogenic factor) in a group of untrained men aged 50-60 years with obesity. METHODS: All men were randomised into a "running" group (training 3 times/week, 60 min each, n = 7), a "cycling" group ( training 3 times/week, 90 min each, n = 7) and a sedentary control group ( n = 7). Both training groups worked at moderate intensity (2-4 mmol/l lactate). The intervention had a duration of 6 months. Before and after this period, blood samples were taken from the participants at rest and they underwent a medical investigation. RESULTS: Body mass index (BMI), systolic and diastolic blood pressure, and plasma levels of VEGF and endostatin were comparable in all three groups. Endurance training significantly reduced BMI in both exercise groups (mean (SEM) before v after 29.7 (0.7) v 29.1 (0.6) kg/m2 and 31.1 (0.7) v 30.1 (0.9) kg/m2 for the running and cycling groups respectively) but not in the control group (30.0 (1.0) v 30.2 (0.8) kg/m2). Endurance training did not influence VEGF plasma level (before v after 1.3 (0.4) v 1.5 (0.2) ng/ml for the running group; 1.6 (0.3) v 1.5 (0.2) ng/ml for the cycling group; and 2.5 (0.6) v 2.1 (0.7) ng/ml for the control group). Plasma level of endostatin was significantly reduced in both exercise groups (mean (SEM) before v after: 20.9 (1.6 v 17.5 (1.0) ng/ml and 21.3 (1.4 v 18.0 (1.6) ng/ml for the running and cycling groups respectively) but not in controls (19.7 (1.3 v 17.7 (1.1 ng/ml). CONCLUSIONS: Endurance training may reduce the antiangiogenic mechanisms in men aged 50-60 years by reducing endostatin plasma level and this may subsequently decrease the risk of cardiovascular disease.

Cross-priming is under control of the relB gene.

Cross-priming is an important mechanism of intercell transfer of antigenic material leading to the specific activation of cytotoxic T lymphocytes. Dendritic cells (DCs) are considered the central antigen-presenting cell in cross-priming. Here we decided to probe the role of the relB gene, a regulator of DC differentiation, in the in vivo cross-priming of a model tumour antigen, TAP(-/-) murine embryo cells (MEC), expressing human adenovirus type 5 early region 1. To this end, we used relB(-/-) mutant mice to generate bone marrow (BM) chimeras as these possess few residual DC but are capable of initiating CD4+ and CD8+ T-cell responses in vivo. Our results show that relB(-/-) BM chimeras are unable to cross-prime CD8+ T cells, suggesting that the relB gene regulates cross-priming.

Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells.

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1-/-) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.

Stimulation via CD40 can substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus.

Reactivation of latent herpesviruses is a particular problem in immunocompromised individuals, such as AIDS patients, who lack effective CD4 T helper cell function. An important question is whether residual immune defenses can be mobilized to combat such opportunistic infections, in the absence of CD4 T cells. In the present study, we used a mouse model of opportunistic infection to determine whether stimulation via CD40 could substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus. Treatment with an agonistic antibody to CD40 was highly effective in preventing reactivation of latent murine gammaherpesvirus (MHV-68) in the lungs of CD4 T cell-deficient mice. CD8(+) T cells were essential for this effect, whereas virus-specific serum antibody was undetectable and IFN-gamma production was unchanged. This demonstration that immunostimulation via CD40 can replace CD4 T cell help in controlling latent virus in vivo has potential implications for the development of novel therapeutic agents to prevent viral reactivation in immunocompromised patients.

Naïve CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation.

In defense of the host, the immune system must often raise an effective cytotoxic T lymphocyte (CTL) response from a small number of clonal precursors. The degree to which activation stimuli regulate the expansion and differentiation of naïve CTLs, however, remains unknown. Using an engineered antigen-presenting cell (APC) system that allows control over antigenic stimulation, we studied the signaling duration requirements for priming and clonal expansion of naïve CTLs. We found that naïve CTLs become committed after as little as 2 h of exposure to APCs and that their subsequent division and differentiation can occur without the need for further antigenic stimulation of the daughter cells, whether priming is in vitro or in vivo. These data show that after a brief interaction with stimulatory APCs, naïve CTLs initiate a program for their autonomous clonal expansion and development into functional effectors.

The roles of MHC class II, CD40, and B7 costimulation in CTL induction by plasmid DNA.

DNA-based vaccines generate potent CTL responses. The mechanism of T cell stimulation has been attributed to plasmid-transfected dendritic cells. These cells have also been shown to express plasmid-encoded proteins and to become activated by surface marker up-regulation. However, the increased surface expression of CD40 and B7 on these dendritic cells is insufficient to overcome the need for MHC class II-restricted CD4(+) T cell help in the priming of a CTL response. In this study, MHC class II(-/-) mice were unable to generate a CTL response following DNA immunization. This deficit in CTL stimulation by MHC class II-deficient mice was only modestly restored with CD40-activating Ab, suggesting that there were other elements provided by MHC class II-restricted T cell help for CTL induction. CTL activity was also augmented by coinjection with a vector encoding the costimulatory ligand B7.1, but not B7.2. These data indicate that dendritic cells in plasmid DNA-injected mice require conditioning signals from MHC class II-restricted T cells that are both CD40 dependent and independent and that there are different roles for costimulatory molecules that may be involved in inducing optimal CTL activity.

CD40 signaling replaces CD4+ lymphocytes and its blocking prevents chronic rejection of heart transplants.

Chronic rejection remains the major obstacle to long term survival in heart transplant recipients. The cellular and molecular mechanisms that underlie chronic rejection are not known, and their discovery can form the basis of clinical intervention. Several investigators have suggested that the development of chronic rejection in solid organ transplants is dependent on help mediated by CD4(+) lymphocytes. Importantly, the mechanism through which help is provided has not been fully delineated in transplant rejection. Using a murine heterotopic heart transplant model without immunosuppression, this study defines the functional role of CD4(+) lymphocytes in chronic rejection. In an MHC class II-mismatched model, we demonstrate that chronic rejection was absolutely contingent on the presence of CD4(+) lymphocytes. Importantly, here we report that signaling through CD40 can replace the requirement of CD4(+) lymphocytes, demonstrated by the development of chronic rejection in CD4 knockout recipients treated with a CD40-activating mAb (FGK45). The return of rejection appears to be a CD8(+) lymphocyte-dependent process, noted by the absence of rejection in FGK45-treated recombinase-activated gene knockout (CD4(+) and CD8(+) lymphocyte-deficient) recipients. The CD40 signaling pathway works independently of B7-CD28 costimulation, as indicated by the development of severe chronic rejection in CD28 knockout recipients. Importantly, this study provides evidence that CD40 ligand-targeted therapies may prevent chronic rejection only in strain combinations where CD4(+) lymphocyte help is absolutely required.

Functional cooperation between T helper cell determinants.

The immune response to T helper (Th) cell determinants of a variety of antigens is often poor and limits severely the potential efficacy of current therapeutic measures through vaccination. Here, we report that an immunologically silent tumor determinant can be rendered immunogenic if linked with a dominant determinant of a parasite antigen, suggesting the existence of functional Th-Th cooperation in vivo. This phenomenon could be mimicked in part by signaling either through CD40 to the antigen-presenting cells or through OX40 to the tumor-determinant reactive T cells, with maximal effects obtained by combined anti-CD40 and anti-OX40 treatment in vivo. The data suggest that CD4 T cells reactive with a dominant determinant provide help to other CD4 T cells through up-regulating the costimulatory ability of antigen-presenting cells, in much the same way as help for CD8 cells. CD4 help for CD4 T cells represents a new immunological principle and offers new practical solutions for vaccine therapy against cancer and other diseases in which antigenic help is limiting.

Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis.

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I-restricted re-presentation of gp96-associated peptides and CTL activation; non-receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

Role of antigen-presenting cells in mediating tolerance and autoimmunity.

The mechanisms that determine whether receptor stimulation leads to lymphocyte tolerance versus activation remain poorly understood. We have used rat insulin promoter (RIP)-gp/P14 double-transgenic mice expressing the lymphocytic choriomeningitis virus (LCMV) glycoprotein (gp) on pancreatic beta-islet cells together with T cells expressing an LCMV-gp-specific T cell receptor to assess the requirements for the induction of autoimmunity. Our studies have shown that administration of the gp peptide gp33 leads to the activation of P14-transgenic T cells, as measured by the upregulation of activation markers and the induction of effector cytotoxic activity. This treatment also leads to expansion and deletion of P14 T cells. Despite the induction of cytotoxic T lymphocyte activity, peptide administration is not sufficient to induce diabetes. However, the administration of gp peptide together with an activating anti-CD40 antibody rapidly induces diabetes. These findings suggest that the induction of tolerance versus autoimmunity is determined by resting versus activated antigen-presenting cells.

Melanoma immunotherapy by targeted IL-2 depends on CD4(+) T-cell help mediated by CD40/CD40L interaction.

The induction of tumor-protective immunity against malignancies remains a major challenge in cancer immunotherapy. A novel, humanized anti-ganglioside-GD(2)-IL-2 immunocytokine (hu14.18-IL-2) induced CD8(+) T cells to eradicate established pulmonary metastases of B78-D14 murine melanoma, in a process that required help by CD4(+) T cells and was mediated by the CD40/CD40 ligand (CD40L) interaction. The anti-tumor effect was diminished in mice deficient in CD4(+) T-cells. Three lines of evidence show that CD4(+) T-cell help was mediated by CD40/CD40L interaction but not by endogenous IL-2 production. First, the hu14.18-IL-2-induced anti-tumor response is partially abrogated in C57BL/6J CD40L knockout (KO) mice in contrast to C57BL/6J IL-2 KO animals, in which the immunocytokine was completely effective. Second, partial abrogation of the anti-tumor effect is induced with anti-CD40L antibodies to the same extent as with CD4(+) T-cell depletion. Third, a complete anti-tumor response induced by hu14.18-IL-2 can be reconstituted in C57BL/6J CD40L KO mice by simultaneous stimulation with an anti-CD40 mAb. These results suggest that help provided by CD4(+) T cells via CD40/CD40L interactions in our tumor model is crucial for effective immunotherapy with an IL-2 immunocytokine.

CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.

The outcome of antigen recognition by naive CD8+ cytotoxic T lymphocytes (CTLs) in the periphery is orchestrated by CD4+ T-helper cells, and can either lead to priming or tolerization. The presence of T-helper cells favors the induction of CTL immunity, whereas the absence of T-helper cells can result in CTL tolerance. The action of T helper cells in CTL priming is mediated by CD40-CD40 ligand interactions. We demonstrate here that triggering of CD40 in vivo can considerably enhance the efficacy of peptide-based anti-tumor vaccines. The combination of a tolerogenic peptide vaccine containing a minimal essential CTL epitope with an activating antibody against CD40 converts tolerization into strong CTL priming. Moreover, CD40 ligation can provide an already protective tumor-specific peptide vaccine with the capacity to induce therapeutic CTL immunity in tumor-bearing mice. These findings indicate that the CD40-CD40 ligand pair can act as a 'switch', determining whether naive peripheral CTLs are primed or tolerized, and support the clinical use of CD40-stimulating agents as components of anti-cancer vaccines.

Conversion of tumor-specific CD4+ T-cell tolerance to T-cell priming through in vivo ligation of CD40.

Tumor antigen-specific T-cell tolerance limits the efficacy of therapeutic cancer vaccines. Antigen-presenting cells mediate the induction of T-cell tolerance to self-antigens. We therefore assessed the fate of tumor-specific CD4+ T cells in tumor-bearing recipients after in vivo activation of antigen-presenting cells with antibodies against CD40. Such treatment not only preserved the responsiveness of this population, but resulted in their endogenous activation. Established tumors regressed in vaccinated mice treated with antibody against CD40 at a time when no response was achieved with vaccination alone. These results indicate that modulation of antigen-presenting cells may be a useful strategy for enhancing responsiveness to immunization.

Ehlers-Danlos syndrome type IV with Charcot Marie tooth disease: an unusual combination of diseases.

Ehlers-Danlos Syndrome (EDS) type IV is a rare genetic disorder of connective tissue. Most patients with EDS type IV are frequently unaware of this disorder until the catastrophic rupture of an artery or bowel occurs. We are reporting an association between this and another uncommon autosomal dominant disorder, Charcot Marie Tooth disease. The neurologic problem led to painful foot deformities, requiring surgery, which was complicated by difficulty controlling bleeding in the friable tissues. Other reported associations of heritable disorders of connective tissue and neuropathies are described.

CD40-CD40Ligand interactions and their role in cytotoxic T lymphocyte priming and anti-tumor immunity.

Tumor-specific immunity relies on interactions with the antigen receptors as well as costimulatory molecules, such as those of the CD28/B7 pathway and relatives of the TNFR gene family. Cytotoxic T lymphocytes specific for cellular antigens are in general primed by professional antigen-presenting cells that indirectly present antigens derived from cells in the periphery. This cross-priming of CD8(+) T cells requires signals provided by CD4(+) T helper cells. Although this dependency on [help' for efficient cytotoxic T lymphocyte priming has been well documented, it was only recently that more mechanistic insight into the nature of this event has been obtained. In the absence of the CD4(+) T cells, signalling through CD40 can replace 'help' required for priming of these CD8(+) T cells. These observations indicate that T cell help for cytotoxic T lymphocytes is mediated by CD40-CD40Ligand (L) interactions, most likely through activation of professional antigen-presenting cells that cross-present cellular antigens to these T cells.

Cross-priming of CTL responses in vivo does not require antigenic peptides in the endoplasmic reticulum of immunizing cells.

It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1-/- cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.

Cryptic open reading frames in plasmid vector backbone sequences can provide highly immunogenic cytotoxic T-lymphocyte epitopes.

Murine tumor cells obtained through transfection of expression plasmids carrying activated cellular and/or viral oncogenes constitute formidable tools for immunological tumor research. As reported previously, mouse embryo cells of C57BL/6 origin, transformed by mutated p53 or human papilloma virus type 16 (HPV16), present, at their surface, MHC-bound peptides that are derived from the p53 and the HPV16 E7 oncoproteins, respectively, which can serve as a target for a highly effective antitumor T-cell response. Here, we describe the identification, through molecular cloning, of an additional, highly immunodominant peptide that is presented by the aforementioned HPV16- and p53-transformed cells. This peptide is encoded by a cryptic open reading frame in the backbone sequences of the plasmids that had been used to generate these cells. Considerable amounts of transcripts encompassing this open reading frame were detected in the cells concerned. These transcripts were the result of the bidirectional nature of the retroviral long terminal repeat (LTR) present in the expression plasmids used for transfection, which resulted in transcription of the gene of interest, as well as in transcription of the vector sequences positioned at the other side of the LTR. Due to this mechanism, all tumor cells harboring LTR-driven expression plasmids expressed the highly immunogenic peptide, whereas cells containing plasmids driven by more unidirectional promoters exhibited lower levels of this peptide. LTR-driven expression plasmids were also shown to encode this peptide epitope when used for DNA vaccination, as mice vaccinated with such a plasmid developed a CTL response against this peptide. Our data show that awareness of plasmid backbone-derived epitopes is of crucial importance for the correct interpretation of preclinical experiments and for the design of DNA vaccines.

Efficient direct priming of tumor-specific cytotoxic T lymphocyte in vivo by an engineered APC.

Although numerous studies have documented a role for B7-1 (CD80) in the induction of antitumor CTL immunity, it is presently unclear to what extent expression of this costimulatory molecule truly endows tumors with significant in vivo APC (antigen-presenting cell) capacity. Recent studies have, in fact, demonstrated that cross-priming, rather than direct priming, may constitute the major mechanism of CTL induction by B7-1 expressing tumors. We have, therefore, investigated the requirements for antigen density and costimulatory molecules in direct CTL priming with a prototype cell-based vaccine that uses a signal sequence-containing minigene to direct expression of a tumor-specific CTL epitope to the endoplasmic reticulum. This design limits sources of antigen available to professional APC in the host and, thereby, the contribution of cross-priming. Induction of antitumor CTL immunity by our prototype APC was shown to solely involve direct priming, independent of host APC, NKI.1+ cells, and CD4+ T cell help. CTL induction through this mechanism required the engineered APC to express the B7-1 molecule as well as a sufficiently high density of peptide/MHC complexes at its surface. Our data, in contrast to previous studies using modified tumor cells, clearly define the antigenic and costimulatory requirements for a suitably engineered "artificial" APC to directly prime peptide-specific CTL in vivo, and demonstrate that the signal sequence minigene approach allows the engineering of highly effective and well-defined cellular vaccines for activation of CTL against epitopes of choice.