Manufacturing of cellular products for therapeutic purposes like stem cell or cancer therapy requires equipment with specific characteristics not always addressed by conventional technologies. A new integrated cell processing device is... more
Manufacturing of cellular products for therapeutic purposes like stem cell or cancer therapy requires equipment with specific characteristics not always addressed by conventional technologies. A new integrated cell processing device is presented that can handle all current technical requirements for manufacturing cellular products by automation of the complete process in a GMP-compliant single-use tubing set. Its capabilities are exemplified in the presented study by successful processing of adult stem cells, natural killer cells, and several cell lines. Multiple cell processing workflows can be automated in a functionally closed environment: from cell separation through cell culture to formulation of the final product.
Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal... more
Anergy is a major mechanism to ensure antigen-specific tolerance in T lymphocytes in the adult. In vivo, anergy has mainly been studied at the cellular level. In this study, we used the T-cell-activating superantigen staphylococcal enterotoxin A (SEA) to investigate molecular mechanisms of T-lymphocyte anergy in vivo. Injection of SEA to adult mice activates CD4+ T cells expressing certain T-cell
A series of novel chemically defined soluble oligomers of the human MHC class II protein HLA-DR1 was constructed to probe the molecular requirements for initiation of T cell activation. MHC dimers, trimers, and tetramers stimulated T... more
A series of novel chemically defined soluble oligomers of the human MHC class II protein HLA-DR1 was constructed to probe the molecular requirements for initiation of T cell activation. MHC dimers, trimers, and tetramers stimulated T cells, as measured by upregulation of the activation markers CD69 and CD25, and by internalization of activated T cell receptor subunits. Monomeric MHC-peptide complexes
Bcl10 is a critical regulator of NF-kappa B activity in T and B cells, coupling antigen receptor signaling to NF-kappa B activation via protein kinase C (PKC). Here we show that PKC or T-cell receptor (TCR)/CD28 signaling results in... more
Bcl10 is a critical regulator of NF-kappa B activity in T and B cells, coupling antigen receptor signaling to NF-kappa B activation via protein kinase C (PKC). Here we show that PKC or T-cell receptor (TCR)/CD28 signaling results in downregulation of Bcl10 protein levels, thereby attenuating NF-kappa B transcriptional activity. Bcl10 degradation requires an intact caspase recruitment domain and is not observed after stimulation with tumor necrosis factor alpha or lipopolysaccharides. Bcl10 downregulation is not affected by proteasome inhibitors but is accompanied by transient localization to lysosomal vesicles, suggesting involvement of the lysosomal pathway rather than the proteasome. The HECT domain ubiquitin ligases NEDD4 and Itch promote ubiquitination and degradation of Bcl10, thus downmodulating NF-kappa B activation. Since CD3/CD28-induced activation of JNK is not affected by the decline of Bcl10, degradation of Bcl10 selectively terminates IKK/NF-kappa B signaling in respons...
Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent... more
Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent upon which anti-CD3 mAb was used as the stimulus, being more pronounced when the anti-CD3 mAb 454 was used as the stimulus than when either anti-CD3 mAb 147 or 446 was the stimulus. The effects of neuraminidase-treated sheep erythrocytes (which bind to CD2) were also more pronounced on mAb 454-induced proliferation than on mAb 147- or 446-induced proliferation. Furthermore, the effects of preincubation with anti-CD2 mAb depended upon the responder status of the donor to IgG1 anti-CD3 mAb. Preincubation of high-responder cells with anti-CD2 mAb had little effect on subsequent IgG1 anti-CD3-induced proliferation. In contrast, preincubation of low-responder cells with anti-CD2 mAb usually augmented the otherwise small proliferative response to IgG1 anti-C...
T cells are activated by binding of the T cell receptor (TCR) to a peptide-major histocompatibility complex (MHC) complex (pMHC) expressed on the surface of antigen presenting cells. Various models have predicted that activation is... more
T cells are activated by binding of the T cell receptor (TCR) to a peptide-major histocompatibility complex (MHC) complex (pMHC) expressed on the surface of antigen presenting cells. Various models have predicted that activation is limited to a narrow window of affinities (or dissociation rates) for the TCR–pMHC interaction and that above or below this window, T cells will fail to undergo activation. However, to date there have not been TCRs with sufficiently high affinities in order to test this hypothesis. In this report we examined the activity of a CD8-negative T cell line transfected with a high affinity mutant TCR (KD = 10 nM) derived from cytotoxic T lymphocyte clone 2C by in vitro engineering. The results show that despite a 300-fold higher affinity and a 45-fold longer off-rate compared with the wild-type TCR, T cells that expressed the mutant TCRs were activated by peptide. In fact, activation could be detected at significantly lower peptide concentrations than with T cell...