Mitomycin C-treated dendritic cells inactivate autoreactive T cells: toward the development of a tolerogenic vaccine in autoimmune diseases.

Treatment of autoimmune diseases remains a challenge for immunological research. An ideal therapy should inhibit the immune reaction against the diseased organ and leave the rest of the immune response intact. Our previous studies showed that donor-derived dendritic cells (DCs) treated in vitro with mitomycin C (MMC) suppress rat heart allograft rejection if injected into recipients before transplantation. Here we analyze their efficacy in controlling autoimmunity. MMC-DCs loaded with myelin-basic-protein (MBP) inhibited specific T cells derived from multiple sclerosis patients in vitro. If coincubated with MMC-DCs, T cells were arrested in the G(0)/G(1) cell cycle phase. Microarray gene scan showed that MMC influences the expression of 116 genes in DCs, one main cluster comprising apoptotic and the second cluster immunosuppressive genes. Apparently, the combination of apoptosis with expression of tolerogenic molecules renders MMC-DCs suppressive. MBP-loaded MMC-DCs also inhibited mouse T cells in vitro and, in contrast to MBP-loaded naïve DCs, did not induce experimental autoimmune encephalitis. Most importantly, mice vaccinated with inhibitory DCs became resistant to the disease. Whereas this is not the first report on generation of suppressive DCs, it delineates a method using a clinically approved drug at nontoxic concentrations, which yields irreversibly changed DCs, effective across species in vitro and in vivo.

No evidence for PERV release by islet cells from German landrace pigs.

BACKGROUND: Islet cells from pig could be used as an alternative to the current treatment of diabetic patients. However, xenotransplantation from pig to humans may be associated with the risk of transmission of porcine endogenous retroviruses (PERVs) that are present in the genome of all pigs and infect human cells in vitro. Although transplantation of pig islet cells for treatment of diabetes may be not accompanied by immunosuppression that may facilitate virus survival, since islets will be used encapsulated, it is nevertheless of importance to study whether islet cells release PERVs able to infect human cells during co-incubation. MATERIAL/METHODS: Isolated islets from German landrace pigs were incubated with highly susceptible human 293 cells for one week. In order to prevent microchimerism 293 cells were made neomycin-resistant (293(neo+)), that allows the elimination of pig cells by a selection medium. The infection of 293(neo+ )target cells was analysed by PCR using PERV-specific primers up to fi ve weeks after co-cultivation. In addition, expression of viral mRNA in pig islet cells was studied by RT-PCR analysis, the expression of viral protein by FACS analysis. RESULTS: Despite the presence of numerous PERV proviruses in the genome of all pigs, no expression of PERV was observed in German landrace pig islet cells, neither as mRNA, nor as protein, nor as viral particles. CONCLUSIONS: Islet cells from German landrace pigs do not express PERVs and may therefore be used for breeding genetically modified pigs suitable for xenotransplantation and treatment of diabetes.

Enhanced T-cell activation and T-cell-dependent IL-2 production by CD83+, CD25high, CD43high human monocyte-derived dendritic cells.

Although standardized protocols are widely used for the generation of monocyte-derived immunostimulatory dendritic cells (DC(ims)), the inducibility of Th1 cells by DC(ims) may considerably differ. As a measure for the quality of DC(ims) generated from an individual donor at a certain time point, CD83 is used in combination with HLA-DR and CD86 to assess DC maturation. When phenotypically analyzing DC(ims), we identified a subpopulation ( approximately 60%) of CD83+, CD86+, and HLA-DR+ DC(ims) that co-expressed CD25. DC within a given DC(ims) preparation identified by lower expression of CD83 and by selective expression of CD14, however, did not co-express CD25. In order to establish CD25 as an additional maturation marker of DC(ims), we studied the DC phenotype of these cells as well as the DC-dependent T-cell proliferation and T-cell cytokine production profile after co-incubation with sorted CD25(high) and CD25(low) subpopulations of CD83+, HLA-DR+, CD86+ DC(ims). CD25(high) DC(ims) showed significant up-regulation of the DC activation molecule CD43 and induced increased levels of IL-2 secretion in allogeneic T-cells (170.7+/-86.7pg/mL) as compared to T-cells coincubated with CD25(low) DC(ims) (86.6+/-37.6pg/mL) [p=0.0224]. This was reflected by a significantly lower T-cell stimulatory capacity of CD25(low) DC(ims) (84.0% of CD25(high) DC(ims), 1:10 ratio; p=0.014) whereas the T-cell stimulatory capacity of CD25(low) DC(ims) was much higher when compared to IL-10 induced regulatory DC (55.3% of CD25(high) DC(ims); 1:10 ratio). With regard to cancer vaccination protocols, we propose to use CD25 and CD43 as additional markers for DC quality control, assessment of maturational status, and positive selection.

Immunosuppressive agents mediate reduced allostimulatory properties of myeloid-derived dendritic cells despite induction of divergent molecular phenotypes.

Immunosuppressive drugs such as glucocorticoids (dexamethasone (Dexa)), cyclosporin A (CsA) and tacrolimus (Tacro) have been shown to impair differentiation and/or function of immunostimulatory dendritic cells (DC(ims)). Phenotypes and functions of the resultant myeloid dendritic cells, however, have not yet been thoroughly elucidated. We show here that all DC subsets generated by treatment with immunosuppressive agents exhibited considerably reduced allostimulatory properties as measured in the primary mixed lymphocyte reaction (tacrolimus>cyclosporin A>dexamethasone, used at equimolar concentrations). In the MLR, all these DC subsets furthermore inhibited secretion of the T-helper type 1 cytokine IFN-gamma; in addition, DC-Tacro and, less so, DC-CsA induced the T-helper type 2 cytokine IL-4. Upon FACS analysis, DC-Tacro and DC-CsA exhibited phenotypic features similar to DC(ims). In addition, DC-CsA and, to a smaller extent, DC-Tacro were characterized by increased mRNA expression of the novel costimulatory molecule B7-H2 (ICOS-ligand). In contrast, dexamethasone induced the generation of DC characterized by decreased expression of CD83 and CD86, by de novo expression of plasmacytoid and myeloid cell markers CD123 and CD14, respectively, and by sustained expression of Toll-like receptor 2. Interestingly, activation of DC-Dexa with a specific TLR2 ligand induced a strong up-regulation of IL-10 along with TNF-alpha and IL-6, a combination of cytokines that allow amplification of regulatory DC populations. In conclusion, myeloid DC induced by dexamethasone as well as by CsA or tacrolimus show reduced allostimulatory properties; however, they are equipped with different molecular repertoires to exert these functions.

A gene signature of inhibitory MHC receptors identifies a BDCA3(+) subset of IL-10-induced dendritic cells with reduced allostimulatory capacity in vitro.

Dendritic cells (DC) are crucial gatekeepers in regulating immunity. Whereas mature immunostimulatory myeloid DC (DC(ims)) potently promote immune responses, IL-10-induced myeloid DC (DC-IL-10) counteract T cell activation. To elucidate the molecular repertoire by which DC-IL-10 secure reduced T cell activation, comparative gene expression profiling was done using Affymetrix U133A microarrays. Among the genes overexpressed in DC-IL-10, eight immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing inhibitory molecules (ILT2, ILT3, ILT4, ILT5, DCIR, PILRA, FcgammaRIIB, SLAM) were found. Phenotypic analysis of DC-IL-10 defined an ILT(high) DC subset further characterized by expression of CD14, TLR2, DC-SIGN, and CD123 and the lack of lymphocyte, monocyte/macrophage, and plasmacytoid DC markers such as CD3, CD8, and CD68. A unique feature of the ILT(high) DC subset was expression of the novel DC marker BDCA3, which was not detected on monocytes, immature DC, DC(ims), or ILT(low) DC-IL-10. While the allogeneic T cell proliferation induced by the entire DC-IL-10 population was approximately 30% of that stimulated by DC(ims), allogeneic MLR responses driven by the ILT(high) DC subset were reduced to 8% of the allostimulatory capacity of DC(ims), although secretion of the inhibitory cytokine IL-10 and other Th1/Th2-associated cytokines was similar in these cells.

Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues.

MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mphi2), and the hepatic sinusoidal endothelial hyaluronan clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18-20 epidermal-growth-factor domains, one X-link domain and three to six B-(X(7))-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mphi2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mphi2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mphi2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell-cell and cell-matrix interactions in vascular function and inflammatory processes.