Mesenchymal stromal cells inhibit proliferation of virus-specific CD8(+) T cells.

Mesenchymal stromal cells (MSCs) possess broad immunomodulatory capacities that are currently investigated for potential clinical application in treating autoimmune disorders. Third-party MSCs suppress alloantigen-induced proliferation of peripheral blood mononuclear cells providing the rationale for clinical use in graft-versus-host disease (GvHD). We confirmed that MSCs strongly inhibited proliferation of CD8(+) T cells in a mixed lymphocyte reaction. However, MSCs also suppressed proliferation of T cells specifically recognizing cytomegalovirus (CMV) and influenza virus. Inhibition was dose dependent, but independent of the culture medium. MSCs inhibited proliferation of specific CD8(+) T cells and the release of IFN-γ by specific CD8(+) T cells for immunodominant HLA-A2- and HLA-B7- restricted antigen epitopes derived from CMV phosphoprotein 65 and influenza matrix protein. This is in contrast to a recently reported scenario where MSCs exert differential effects on alloantigen and virus-specific T cells potentially having an impact on surveillance and prophylaxis of patients treated by MSCs.

Evidence for radiosensitizing by gliotoxin in HL-60 cells: implications for a role of NF-kappaB independent mechanisms.

Radioresistance markedly impairs the efficacy of tumor radiotherapy and may involve antiapoptotic signal transduction pathways that prevent radiation-induced cell death. A common cellular response to genotoxic stress induced by radiation is the activation of the nuclear factor kappa B (NF-kappaB). NF-kappaB activation in turn can lead to an inhibition of radiation-induced apoptotic cell death. Thus, inhibition of NF-kappaB activation is commonly regarded as an important strategy to abolish radioresistance. Among other compounds, the fungal metabolite gliotoxin (GT) has been reported to be a highly selective inhibitor of NF-kappaB activation. Indeed, low doses of GT were sufficient to significantly enhance radiation-induced apoptosis in HL-60 cells. However, this effect turned out to be largely independent of NF-kappaB activation since radiation of HL-60 cells with clinically relevant doses of radiation induced only a marginal increase in NF-kappaB activity, and selective inhibition of NF-kappaB by SN50 did not result in a marked enhancement of GT-induced apoptosis. GT induced activation of JNKs, cytochrome c release from the mitochondria and potently stimulated the caspase cascade inducing cleavage of caspases -9, -8, -7 and -3. Furthermore, cleavage of the antiapoptotic protein X-linked IAP and downregulation of the G2/M-specific IAP-family member survivin were observed during GT-induced apoptosis. Finally, the radiation-induced G2/M arrest was markedly reduced in GT-treated cells most likely due to the rapid induction of apoptosis. Our data demonstrate that various other pathways apart from the NF-kappaB signaling complex can sensitize tumor cells to radiation and propose a novel mechanism for radiosensitization by GT, the interference with the G2/M checkpoint that is important for repair of radiation-induced DNA damage in p53-deficient tumor cells.

Molecular cloning and characterization of the human protein kinase D2. A novel member of the protein kinase D family of serine threonine kinases.

We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKCmicro and PKCnu, particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase domain. In contrast, it shows only a low degree of sequence similarity to other members of the PKC family. Therefore, the new protein has been termed protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various human cell lines, including HL60 cells, which do not express PKCmicro. In vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [(3)H]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser(876) residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser(876) of PKD2 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCK(B)/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.

Thymectomy and azathioprine have no effect on the phenotype of CD4 T lymphocyte subsets in myasthenia gravis.

The influence of thymectomy and long term immunosuppression on the phenotype of CD4 T lymphocyte subsets, which were defined by the restricted expression of CD45RA and CD45RO markers, was studied by double immunofluorescence in 29 patients in different clinical stages of generalised myasthenia gravis. In the acute stage of myasthenia, before thymectomy and immunosuppression, no differences in CD4 subsets were observed in the peripheral blood from nine patients and 21 matched controls. Four to seven weeks after thymectomy, there was a slightly decreased proportion of CD4+CD45RO+ (UCHL1+) memory cells (p < 0.05, paired t test). Patients on steroids showed a more pronounced decrease of CD4+CD45RO+ cells suggesting, in addition, a drug-related effect. CD4 subsets (CD45RA, CD45RO, and CD29 positive) in the peripheral blood compartment remained largely stable over 18 to 24 months thereafter. In addition, CD4 subsets were examined in 20 patients with myasthenia gravis who had had a thymectomy between two and 17 years before. With the exception of patients on steroids, there were no differences in CD4 subsets in patients on or off azathioprine. These data did not show any relation of CD4 T cell subsets to the clinical course of myasthenia, or significant changes due to thymectomy, or immunosuppression with azathioprine. These results also complement the authors' clinical experience that thymectomy in adults does not leave a deficit in cell-mediated immunity. The slight change associated with steroid treatment might deserve further attention.

T cells from normal and myasthenic individuals recognize the human acetylcholine receptor: heterogeneity of antigenic sites on the alpha-subunit.

The alpha-subunit of the nicotinic acetylcholine receptor is the major target of the autoimmune response in myasthenia gravis. We investigated the proliferative response of T cells from patients with myasthenia gravis and healthy volunteers to recombinant polypeptides of the human acetylcholine receptor including the full-length alpha-subunit (alpha 1-437). T cells from 20 (71%) of 28 patients and 7 (37%) of 19 healthy volunteers responded in primary cultures. Subsequently, specific T-cell lines were established: CD4+, CD8-, UCHL1+, and major histocompatibility complex (MHC) class II-restricted. Using a set of fragments of the alpha-subunit, major antigenic sites could be localized on the extracellular, N-terminal part of the molecule as well as close to the C-terminus. The T-cell response was heterogeneous, both among different individuals and among T-cell lines from a single donor. These T cells did not cross-react with Torpedo acetylcholine receptor, which was previously used as a substitute for human muscle acetylcholine receptor, suggesting that the T cells had a bias for unique human sequences. A single antigenic fragment could be presented in the context of different MHC class II molecules, and different fragments could be presented in the context of the same MHC molecule. This supports earlier observations of considerable heterogeneity in dealing with acetylcholine receptor as an autoantigen on the level of both T cells and antigen-presenting cells. The data also demonstrate that acetylcholine receptor-specific T cells are present in the normal immune repertoire, and emphasize the role of immune regulation for maintaining a state of tolerance.