Evidence for a pathogenic role of extrafollicular, IL-10-producing CCR6+B helper T cells in systemic lupus erythematosus.

Interleukin 10 (IL-10) is an antiinflammatory cytokine, but also promotes B cell responses and plays a pathogenic role in systemic lupus erythematosus (SLE). CD4+CCR6+IL-7R+T cells from human tonsils produced IL-10 following stimulation by naïve B cells, which promoted B cell immunoglobulin G (IgG) production. These tonsillar CCR6+B helper T cells were phenotypically distinct from follicular helper T (TFH) cells and lacked BCL6 expression. In peripheral blood, a CCR6+T cell population with similar characteristics was identified, which lacked Th17- and TFH-associated gene signatures and differentiation-associated surface markers. CD4+CCR6+T cells expressing IL-10, but not IL-17, were also detectable in the spleens of cytokine reporter mice. They provided help for IgG production in vivo, and expanded systemically in pristane-induced lupus-like disease. In SLE patients, CD4+CCR6+IL-7R+T cells were associated with the presence of pathogenic anti-dsDNA (double-stranded DNA) antibodies, and provided spontaneous help for autoantibody production ex vivo. Strikingly, IL-10-producing CCR6+T cells were highly abundant in lymph nodes of SLE patients, and colocalized with B cells at the margins of follicles. In conclusion, we identified a previously uncharacterized population of extrafollicular B helper T cells, which produced IL-10 and could play a prominent pathogenic role in SLE.

Protein O-mannosylation is crucial for human mesencyhmal stem cells fate.

Human mesenchymal stem cells (MSC) are promising cell types in the field of regenerative medicine. Although many pathways have been dissected in the effort to better understand and characterize MSC potential, the impact of protein N- or O-glycosylation has been neglected. Deficient protein O-mannosylation is a pathomechanism underlying severe congenital muscular dystrophies (CMD) that start to develop at the embryonic developmental stage and progress in the adult, often in tissues where MSC exert their function. Here we show that O-mannosylation genes, many of which are putative or verified glycosyltransferases (GTs), are expressed in a similar pattern in MSC from adipose tissue, bone marrow, and umbilical cord blood and that their expression levels are retained constant during mesengenic differentiation. Inhibition of the first players of the enzymatic cascade, POMT1/2, resulted in complete abolishment of chondrogenesis and alterations of adipogenic and osteogenic potential together with a lethal effect during myogenic induction. Since to date, no therapy for CMD is available, we explored the possibility of using MSC extracellular vesicles (EVs) as molecular source of functional GTs mRNA. All MSC secrete POMT1 mRNA-containing EVs that are able to efficiently fuse with myoblasts which are among the most affected cells by CMD. Intriguingly, in a pomt1 patient myoblast line EVs were able to partially revert O-mannosylation deficiency and contribute to a morphology recovery. Altogether, these results emphasize the crucial role of protein O-mannosylation in stem cell fate and properties and open the possibility of using MSC vesicles as a novel therapeutic approach to CMD.

Role of antigen-presenting cells in cross-priming of cytotoxic T lymphocytes by apoptotic cells.

Although the mechanisms regulating recognition and phagocytosis of apoptotic cells by scavenger cells are the subject of intense investigation, little is known about the fate of the antigens contained in apoptotic cells and the constraints defining their immunogenicity. We developed a model in C57BL/6 mice to evaluate whether phagocytosis of apoptotic tumor cells yielded antigens able to get access to the MHC class I pathway and activate a specific cytotoxic T lymphocyte response. Our results demonstrate that apoptotic tumor cells are antigenic in vitro and can be immunogenic in vivo. Their immunogenicity depends on the number of cells used for immunization and the antigen-presenting cells involved in processing and presentation of antigens contained in the dying cells. The demonstration of the immunogenicity of apoptotic cells may have direct implications both in autoimmunity and cancer.

Vaccination with mouse mammary adenocarcinoma cells coexpressing B7-1 (CD80) and B7-2 (CD86) discloses the dominant effect of B7-1 in the induction of antitumor immunity.

Nonreplicating TS/A mammary adenocarcinoma cells expressing B7-2 (CD86) (TS/A-2) are more immunogenic than those expressing B7-1 (CD80) (TS/A-1), indicating that B7-1 and B7-2 display nonredundant costimulatory effects in inducing antitumor responses. Whereas transfection of B7-2 cDNA into TS/A-1 cells does not improve their immunogenicity, transfection of B7-1 cDNA into TS/A-2 cells (TS/A-2/1) decreases their immunogenicity in a manner that is directly related to the surface levels of B7-1. Ab blocking of B7-1 on TS/A-2/1 cells before their injection in vivo restores the higher immunogenicity characteristic of single B7-2 transfectants, indicating therefore that B7-1 actively modulates the B7-2-dependent costimulation. The expression of B7-1 also modifies quantitatively the balance of endogenous IFN-gamma and IL-4 induced in vivo by TS/A-2 vaccines. In fact, we find that vaccination with TS/A-2/1 cells results in the production of more IFN-gamma and less IL-4 than TS/A-2 vaccines, a pattern comparable to that induced by TS/A-1 cells. Thus, in the TS/A model of antitumor response, B7-1 modulates B7-2-dependent costimulatory effects in a dominant, noncompetitive way.

Induction of apoptosis by fenretinide (4HPR) in human ovarian carcinoma cells and its association with retinoic acid receptor expression.

We previously reported that fenretinide (4HPR) is effective against a human ovarian carcinoma xenografted in nude mice. The effects of 4HPR on ovarian tumors have been further studied in in vitro ovarian carcinoma cell lines A2780, IGROV-I, SW626 and OVCA432. A2780 was the most sensitive line: 50% growth inhibition was obtained after 3 days of exposure to 1 microM 4HPR, a pharmacologically achievable concentration, whereas approx. 10 microM 4HPR gave a similar inhibition in the other cell lines. All-trans retinoic acid (RA), at doses up to 10 microM, did not inhibit cell proliferation. Gel electrophoresis of DNA from either detached or attached A2780 cells treated with 4HPR revealed DNA ladders in detached cells. Apoptosis was also evidenced in detached 4HPR-treated cells by flow cytometry and microscopic observation. The difference in cell line sensitivity to the anti-proliferative effect of 4HPR was not related to drug uptake or efflux. Only A2780 cells, the most sensitive to 4HPR, expressed constitutive levels of RARbeta; moreover, the levels of RARalpha and RARgamma expression in these cells were higher than in the other cell lines. In A2780 cells, the association of an IC20 of 4HPR to cisplatin resulted in a strong potentiation of the anti-proliferative effect. These data show (i) that 4 HPR, in contrast to RA, has an anti-proliferative effect in human ovarian carcinoma cells which is related to induction of apoptosis and (ii) that among the tested lines, the most responsive to the drug expressed RARbeta and the highest levels of RARalpha and RARgamma. The results also suggest that 4HPR can potentiate the effects of cisplatin in ovarian carcinoma.

Role of retinoic acid receptor overexpression in sensitivity to fenretinide and tumorigenicity of human ovarian carcinoma cells.

The role of retinoic acid receptor (RAR) expression in sensitivity to N-(4-hydroxyphenyl)retinamide (4HPR or fenretinide) as well as on the tumorigenicity of human ovarian carcinoma cells was examined. Two human ovarian cancer cell lines, A2780 and IGROV-1, with a 10-fold difference in sensitivity to 4HPR were chosen to study RAR involvement in the response to 4HPR. To determine which RAR was effective, RARalpha, beta and gamma were individually overexpressed in A2780 cells, which are the most sensitive to 4HPR. Sensitivity to 4HPR was increased in RARbeta-overexpressing clones, whereas it was slightly decreased in RARalpha transfectants (which had diminished RARbeta expression) and was unchanged in clones transfected with RARgamma. IGROV-1 cells, which are RARbeta negative, were transfected with RARbeta. Surprisingly, none of the obtained IGROV-1 RARbeta transfectants expressed RARbeta protein, in spite of RARbeta mRNA transcription. All clones were similar to the parental IGROV-1 cells in their sensitivity to 4HPR. Treatment with a pharmacologically achievable concentration of 4HPR (1 microM) led to a rapid 2-fold increase in RARbeta mRNA levels in A2780 cells, but it did not induce RARbeta expression in IGROV-1 cells. Analysis of the tumorigenicity of A2780-transfected clones revealed that overexpression of RARalpha was associated with a significant reduction in tumor takes (50% and 67%, respectively, vs. 96% for the parent line) and with a reduced growth rate. Oncogenicity was clearly decreased in only 1 of the 2 RARbeta-overexpressing clones (33% takes) and was unchanged in the 2 clones with increased RARgamma expression. Our results demonstrate that basal expression and 4HPR inducibility of RARbeta play a role in mediating 4HPR response in ovarian cancer cells. The findings of reduced oncogenicity of clones overexpressing RARalpha and of one clone overexpressing RARbeta indicate that RARalpha and RARbeta might have a tumor-suppressive effect in ovarian tumors.

Bystander apoptosis triggers dendritic cell maturation and antigen-presenting function.

Physiologic cell death via apoptosis occurs without inflammation or autoimmunity. Here, we investigated the outcome of the interaction of apoptotic cells with dendritic cells (DCs), which are potent professional APCs. DCs internalized apoptotic cells and processed them for presentation to both MHC class I- and class II-restricted T cells with an efficiency that was dependent upon the number of apoptotic cells. The latter event was accompanied by the autocrine/paracrine secretion of IL-1beta and TNF-alpha, with eventual DC maturation. High numbers of apoptotic cells, mimicking a failure of their in vivo clearance, are therefore sufficient to trigger DC maturation and the presentation of intracellular Ags from apoptotic cells, even in the absence of exogenous "danger" signals.

Relevance of the tumor antigen in the validation of three vaccination strategies for melanoma.

Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models. Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies. To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model. C57BL/6 mice received one to three s.c. injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture. One to 2 wk later, the animals were challenged s.c. with B16 or B16 cells expressing OVA (B16-OVA). Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA. When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181-188 allowed protection against B16. Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals. Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.

Dendritic cell presentation of antigens from apoptotic cells in a proinflammatory context: role of opsonizing anti-beta2-glycoprotein I antibodies.

OBJECTIVE: To verify whether opsonization of apoptotic cells skews the outcome of apoptotic cell antigen presentation by dendritic cells (DCs). METHODS: RMA cells, which were engineered with a mutant ovalbumin (OVA) protein and were devoid of the leader secretory sequence (OVA-RMA), underwent ultraviolet irradiation to induce apoptosis. Binding of anti-beta2-glycoprotein I antibodies (anti-beta2GPI) and phagocytosis of apoptotic cells were assessed by flow cytometry and confocal microscopy. Presentation of processing antigens and major histocompatibility complex (MHC) class II-restricted or MHC class I-restricted antigens was assessed using OVA-specific T cell hybridomas. RESULTS: Anti-beta2GPI facilitated presentation of epitopes from internalized apoptotic cells to MHC class II-restricted, but not to class I-restricted, T lymphocytes. DCs challenged with supernatants of apoptotic cells did not activate OVA-specific T cells, making it unlikely that anti-beta2GPI complexed with antigen released from dying cells plays a role in antigen presentation. DCs challenged with low numbers of anti-beta2GPI-opsonized apoptotic cells secreted interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-10 in an autocrine/paracrine manner. CONCLUSION: Opsonization influences the outcome of the disposal of low numbers of apoptotic cells by DCs. This implies that soluble factors bound to apoptotic cells modulate their immunogenicity.