Microparticles shed from different antigen-presenting cells display an individual pattern of surface molecules and a distinct potential of allogeneic T-cell activation.

Various cells such as platelets, lymphocytes, endothelial cells, red blood cells and monocytes do release surface-derived microparticles (mps). We analysed mp isolated from supernatants of cultured antigen-presenting human cells (APCs) and human cell lines. Particle sizing by dynamic light scattering revealed a characteristic size of the particles ranging from 80 nm to 300 nm in viable cells and from 400 nm to 1200 nm in irradiated cells. Employing flow-cytometry, we observed partly an altered surface protein composition of the mp compared to their cellular source. Mp originating from dendritic cells (DCs) differed in their surface composition from those released from monocytes and monocyte-derived macrophages. In functional assays, these mp stimulated alloreactive T-cells. The treatment of the cells with either UV-B or lipopolysaccharide strongly influenced the quantity, the immunostimulatory features and the surface composition of the mp. Mp from apoptotic macrophages were able to reduce the stimulatory capacity of vital macrophages but not of DC. Apoptotic mps from DC, on the other hand, were always stimulatory. This is the first report regarding the study of mp released from DC and compared with those released from other APC.

UV irradiation inhibits ABC transporters via generation of ADP-ribose by concerted action of poly(ADP-ribose) polymerase-1 and glycohydrolase.

ATP-binding cassette (ABC) transporters are involved in the transport of multiple substrates across cellular membranes, including metabolites, proteins, and drugs. Employing a functional fluorochrome export assay, we found that UVB irradiation strongly inhibits the activity of ABC transporters. Specific inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) restored the function of ABC transporters in UVB-irradiated cells, and PARP-1-deficient cells did not undergo UVB-induced membrane transport inhibition. These data suggest that PARP-1 activation is necessary for ABC transporter functional downregulation. The hydrolysis of poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase (PARG) was also required, since specific PARG inhibitors, which limit the production of ADP-ribose molecules, restored the function of ABC transporters. Furthermore, ADP-ribose molecules potently inhibited the activity of the ABC transporter P-glycoprotein. Hence, poly(ADP-ribose) metabolism appears to play a novel role in the regulation of ABC transporters.

Apoptosis of the teratocarcinoma cell line Tera-1 leads to the cleavage of HERV-K10gag proteins by caspases and/or granzyme B.

Redistribution, post-translational modifications and coclustering with viral antigens contribute to the immunogenicity of apoptotic cell-derived autoantigens. Almost all known targets of the humoral autoimmune response in systemic lupus erythematosus (SLE) are cleaved by caspases or granzyme B during apoptosis. Antibodies against retroviral proteins can frequently be detected in the sera of SLE patients without overt retroviral infections. These antibodies may represent cross-reactive antibodies or may have been induced by proteins encoded by endogenous retroviral sequences. We used Tera-1 cells that abundantly express a group-specific antigen of human endogenous retroviruses, HERV-K10gag polyprotein, to investigate its processing during apoptosis. Tera-1 cells induced to undergo apoptosis showed an altered HERV-K10gag processing compared with viable cells. In addition, granzyme B was able to cleave HERV-K10gag isolated from viable Tera-1 cells. Similar to nuclear autoantigens, endogenous retroviral proteins are cleaved during the execution phase of apoptosis. These post-translational modifications may result in the generation of T-cell neoepitopes or a changed epitope hierarchy of retroviral proteins. Therefore, immunogenicity of retroviral antigens in SLE patients may result from a similar mechanism as described for nuclear autoantigens.

5,6-carboxyfluorescein diacetate succinimidyl ester-labeled apoptotic and necrotic as well as detergent-treated cells can be traced in composite cell samples.

Detection of dividing cells by staining with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) has been widely used in flow-cytometric protocols. We analyzed the fate of CFSE in cells undergoing apoptotic or necrotic cell death, respectively. Peripheral blood mononuclear cells (PBMC) were stained with CFSE. Apoptosis was induced by UVB irradiation and necrosis by incubation at 56 degrees C for 30 min. In some experiments, labeled cells were permeabilized with detergent and CFSE association with nuclei was assessed. We observed that (i) CFSE remains stably detectable in apoptotic and necrotic cells; (ii) CFSE remains stably associated with the nuclei of cells even after their lysis by detergent; (iii) CFSE labeling does not interfere with the induction of cell death; and (iv) CFSE is not transferred from stained dying cells to unstained neighboring counterparts. We conclude that, in addition to tracking viable cells, CFSE can be used to trace dying cells in composite samples. We demonstrated that CFSE labeling does not influence the induction and the execution of apoptosis or necrosis.

Complement binding is an early feature of necrotic and a rather late event during apoptotic cell death.

The phagocytosis of dying cells is an integral feature of apoptosis and necrosis. There are many receptors involved in recognition of dying cells, however, the molecular mechanisms of the scavenging process remain elusive. The activation by necrotic cells of complement is well established, however, the importance of complement in the scavenging process of apoptotic cells was just recently described. Here we report that the complement components C3 and C4 immediately bound to necrotic cells. The binding of complement was much higher for lymphocytes compared to granulocytes. In case of apoptotic cell death complement binding was a rather late event, which in lymphocytes was preceded by secondary necrosis. Taken together complement binding is an immediate early feature of necrosis and a rather late event during apoptotic cell death. We conclude that complement may serve as an opsonin for fragments of apoptotic cells that have escaped regular scavenging mechanisms.

CD4 positive peripheral T cells from patients with systemic lupus erythematosus (SLE) are clonally expanded.

T cell activation was analysed in peripheral CD4+ T cells from both systemic lupus erythematosus (SLE) patients with active and inactive disease as well as in normal healthy donors (NHD) to investigate the involvement of CD4+ T cells in the etiopathogenesis of SLE. CD4+ T cell receptor (TCR) beta-chain transcripts, containing the complementarity determining region 3 (CDR3), were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and analysed by high-resolution polyacrylamide gel electrophoresis. In addition the CDR3 of both clonally activated as well as heterogeneous Vbeta families from SLE patients were analysed at the molecular level. We observed a restricted CDR3 length polymorphism in peripheral CD4+ T cells from SLE patients compared with NHD, more pronounced in patients with high disease activity. Furthermore, in some Vbeta families single peaks in the histogram indicated nearly monoclonal T cell expansion. Sequencing of selected TCR beta-chains revealed a increased content of acidic amino acids in the CDR3 encoded by either proximal Jbeta elements or N nucleotides. We conclude that CD4+ T cells from peripheral blood of SLE patients display features of a secondary antigen driven immune response. The bias of the CDR3 towards acidic amino acids suggests the involvement of positively charged antigens.

Dominant T cells in idiopathic nephrotic syndrome of childhood.

BACKGROUND: Because of several studies, idiopathic nephrotic syndrome (INS) of childhood is suspected to have an immunologic pathogenesis with T cells playing a major role. To investigate this hypothesis further, we studied the diversity of the CDR3 region of the T-cell receptor (TCR) beta-chain from peripheral T cells isolated from patients with INS. METHODS: The study was performed over a three-year period to obtain longitudinal data on the repertoire of peripheral T cells. mRNA from peripheral mononuclear cells (PBMCs) of seven INS patients and two healthy controls (NHD) was prepared and analyzed for CDR3 length polymorphism of TCR beta-chain by spectratyping. RESULTS: All INS patients presented individually skewed spectratype histograms in at least one Vbeta-family. Patients suffering from a frequent relapsing course of INS or a focal global sclerosis showed some alterations to persist in all samples isolated in the observation period (up to 3 years). In addition, sequence analyses of the beta-chain of the TCR CDR3 region confirmed clonal expansion of peripheral T cells in those patients who had displayed spectratype alterations. CONCLUSIONS: The data give strong evidence for an direct involvement of CD8+ T cells in the complicated course of INS.

Treatment with annexin V increases immunogenicity of apoptotic human T-cells in Balb/c mice.

Exposure of phosphatidylserine on the outer leaflet of the cytoplasmic membrane is an early event during apoptotic cell death and serves as a recognition signal for phagocytes. Usually the clearance of apoptotic cells does not initiate inflammation or immune response. We investigated the immune response in Balb/c mice towards apoptotic human T-cells. Animals injected with apoptotic cells showed significantly reduced humoral immune responses, especially Th1-dependent IgG2a titres, compared to controls immunised with viable cells. However, treatment of apoptotic cells with annexin V (AxV) significantly increased the humoral immune response. AxV binds with high affinity to anionic phospholipids and as a result interferes with the phosphatidylserine recognition by phagocytes. Our results indicate that AxV treatment may be used to increase the efficiency of apoptotic cell-based vaccines, e.g. some tumour vaccines.

Alternatively spliced mRNA molecules of the thrombospondin receptor (CD36) in human PBMC.

We employed reverse transcription polymerase chain reaction (RT-PCR) to detect alternatively spliced CD36 mRNA in human peripheral blood mononuclear cells (PBMC). Sequencing of cloned cDNA revealed alternatively spliced mRNA molecules in 13 out of 39 clones. We observed exon skipping of up to 10 out of 12 coding exons in eight alternative transcripts. Additionally, in five of the transcripts, alternative splice donor or acceptor sites were used during mRNA maturation. Considering the CD36 molecule serves many functions in coagulation, host defence, lipid metabolism, and scavenging, we speculate that the proteins encoded by the alternatively spliced mRNA molecules may be involved in regulation of both CD36 gene expression and function.

Biased TCR repertoire in HIV-1-infected patients due to clonal expansion of HIV-1-reverse transcriptase-specific CTL clones.

To study whether an expansion of HIV-1-specific CTL is contributing to the skewed TCR repertoire in HIV-1-infection, we characterized the TCR usage of CTL clones specific for a conserved epitope in HIV-1 reverse transcriptase (RT/476-484). CTL clones from three HIV-1-infected patients displayed highly similar TCR usage and used the identical Vbeta6.1 and Valpha2.5 gene segments. CTL clones from two patients showed a very high degree of similarity within the TCR complementarity-determining region-3 (CDR-3). In accordance with the similar molecular structure, all three CTL clones also exhibited a similar functional activity with regard to recognition of variant peptides and cytokine secretion pattern. In one subject clonal expansion of a single CTL specificity could be shown over a 10-mo period. TCR spectratyping of PBMC from two patients revealed a marked expansion of CDR-3 segments of a certain length within the Vbeta6-family. Sequence analysis of these CDR-3 yielded sequences identical to the RT/476-484-specific CTL previously isolated from the same patients. This analysis demonstrates that clonal expansion of HIV-1-specific CTL is contributing to the skewed TCR repertoire in HIV-1-infected patients.

Increased spontaneous in vitro apoptosis in double negative T cells of humans with a fas/apo-1 mutation.

We describe a 17 year old patient suffering from Canale-Smith syndrome (CSS) including chronic lymphadenopathy, splenomegaly, hypergammaglobulinemia and recurrent Coombs positive hemolytic crises. The parents are not consanguine, all other family members including two brothers are healthy. Peripheral blood mononuclear cells of the patient showed an increased rate of CD3 positive, CD4/CD8 double negative T-lymphocytes. In vitro assays showed these cells to have an increased rate of spontaneous apoptosis. Though expression of Fas/Apo-1 (CD95) and Fas-ligand (FasL) was detected on RNA- and protein level we found Fas/Apo-1 mediated apoptosis being significantly reduced. Sequencing of the fas/apo-1 gene proved the patient RT and his father to carry a point mutation at position 804 located in exon 9 (death domain) leading to an amino acid substitution. For developing of CSS, a fas/apo-1 mutation seems to be necessary but not sufficient. An additional independent mechanism must be involved in the pathogenesis of human lpr<-phenotype.

Detection of restricted junctional diversity of peripheral T cells in SLE patients by spectratyping.

Analysis of somatic mutations revealed that anti-double-stranded DNA (dsDNA) autoantibodies from patients with systemic lupus erythematosus (SLE) share features of a T cell dependent, antigen driven immune response. Therefore we analysed the length diversity of the complementarity determining region 3 (CDR3) of T cell receptor (TCR) by high resolution gel electrophoresis of 16 V beta family specific RT PCR products (spectratyping). To enable statistical analysis we developed a quantitative scoring method for the histograms. We investigated 16 V beta gene families in peripheral T cells of SLE patients (n = 9) with active (n = 5) and inactive (n = 4) disease as well as normal healthy blood donors (NHD; n = 9). Analysis of TCR V beta spectratypes (active SLE, n = 59; inactive SLE, n = 51 and NHD n = 97) revealed statistically significant differences of CDR3 length distribution between SLE patients and NHD (P < 0.0001 (active SLE/NHD) and P = 0.0034 (inactive SLE/NHD). These results suggest that spectratyping is able to detect clonal activation of peripheral T cells which correlates to disease activity in SLE patients. We conclude that peripheral T cells from SLE patients display features of a secondary antigen driven immune response.