Gijs A. van Seventer

Superantigen-Induced T Cell:B Cell Conjugation Is Mediated by LFA-1 and Requires Signaling Through Lck, But Not ZAP-70

The formation of a conjugate between a T cell and an APC requires the activation of integrins on the T cell surface and remodeling of cytoskeletal elements at the cell-cell contact site via inside-out signaling. The early events in this signaling pathway are not well understood, and may differ from the events involved in adhesion to immobilized ligands. We find that conjugate formation between Jurkat T cells and EBV-B cells presenting superantigen is mediated by LFA-1 and absolutely requires Lck. Mutations in the Lck kinase, Src homology 2 or 3 domains, or the myristoylation site all inhibit conjugation to background levels, and adhesion cannot be restored by the expression of Fyn. However, ZAP-70-deficient cells conjugate normally, indicating that Lck is required for LFA-1-dependent adhesion via other downstream pathways. Several drugs that inhibit T cell adhesion to ICAM-1 immobilized on plastic, including inhibitors of mitogen-activated protein/extracellular signal-related kinase kinase, phosphatidylinositol-3 kinase, and calpain, do not inhibit conjugation. Inhibitors of phospholipase C and protein kinase C block conjugation of both wild-type and ZAP-70-deficient cells, suggesting that a phospholipase C that does not depend on ZAP-70 for its activation is involved. These results are not restricted to Jurkat T cells; Ag-specific primary T cell blasts behave similarly. Although the way in which Lck signals to enhance LFA-1-dependent adhesion is not clear, we find that cells lacking functional Lck fail to recruit F-actin and LFA-1 to the T cell:APC contact site, whereas ZAP-70-deficient cells show a milder phenotype characterized by disorganized actin and LFA-1 at the contact site.

Human helper T cell activation and differentiation is suppressed by porcine small intestinal submucosa.

A cell-free biomaterial derived from porcine small intestinal submucosa (SIS) has been used successfully in many models as a xenogeneic scaffolding material without generating immune-mediated inflammatory reactions. We investigated whether this absence of inflammation is due to the presence of porcine transforming growth factor beta (TGF-beta) activity found in SIS that may have immunosuppressive properties on helper T (Th) cell subset activation and differentiation. We used in vitro models for the generation of human Th1 and Th2 cells to investigate the influence of SIS. We found that SIS partially suppressed Th1 cell expansion and secretion of interleukin 12 (IL-12) and interferon gamma (IFN-gamma) in a TGF-beta-dependent manner, but Th1 cell expansion and IFN-gamma secretion could be fully overcome by addition of recombinant IL-12. The suppression by SIS of Th cell activation also involved the induction of Th cell apoptosis. In addition, SIS completely abolished the generation of Th2 cells in vitro, but this effect of SIS was not reversed by neutralizing TGF-beta antibodies. Our results indicate the presence in SIS of factors that can suppress Th cell activation through both the inhibition of IL-12 secretion and the induction of Th cell apoptosis. We established further that these factors include TGF-beta and at least one other factor.

IL-12 Decreases Activation-Induced Cell Death in Human Naive Th Cells Costimulated by Intercellular Adhesion Molecule-1. I. IL-12 Alters Caspase Processing and Inhibits Enzyme Function

Th cells can receive costimulatory signals through the LFA-1/ICAM-1 accessory pathway that are sufficient to induce early Th cell proliferation, but not subsequent cell expansion and maintenance of cell viability. To investigate the regulatory role for IL-12 in ICAM-1-mediated costimulation, human naive Th cells were stimulated with coimmobilized anti-CD3 mAb and ICAM-1 Ig in the presence or absence of IL-12. The ICAM-1-mediated costimulatory signals in this model resulted in early Th cell proliferation followed by cell death that was partially mediated by Fas and involved loss of mitochondrial membrane potential, processing of procaspase-9 and -3, and activation of caspase-3. Addition of IL-12 prevented activation-induced cell death and promoted late proliferation. ICAM-1 + IL-12-costimulated Th cells were resistant to Fas-mediated cell death through a mechanism that did not appear to involve a decrease in either Fas or Fas ligand expression. IL-12 did not inhibit the loss of mitochondrial membrane potential induced by ICAM-1-mediated costimulation, and this finding was consistent with the inability of IL-12 to increase expression of the antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-xL. Interestingly, IL-12 promoted an altered processing of procaspase-9 and -3 and a decrease in the percentage of cells displaying caspase-3 catalytic function. In conclusion, we now describe a novel regulatory function for IL-12 in preventing Th cell death and, as a result, in greatly increasing Th cell viability and expansion. Together, our findings indicate that IL-12 may perform this regulatory role by preventing Fas-mediated activation-induced cell death through inhibition of caspase-3 enzyme activity.

Pyk2 is differentially regulated by β 1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

β1 integrins can provide T cell co‐stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase‐related tyrosine kinase, is regulated by β1 integrin signaling in human T cells. Stimulation of Jurkat T cells with the α4β1 integrin ligand VCAM‐1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM‐1 and anti‐CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3‐ and α4β1 integrin‐mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3‐derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4+ T cells confirmed the ability of CD3‐derived signals to synergize with β1 integrin‐dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28‐mediated co‐stimulatory signals were able to synergize with CD3‐mediated signals in inducing ERK and JNK activation and secretion of IL‐2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell costimulation mediated specifically by β1 integrins.

Cytokine modulation of costimulatory molecules on human fetal retinal pigment epithelial cells.

Purpose. Experiments were performed to evaluate the effect of various pro- and anti-inflammatory cytokines on the human fetal retinal pigment epitheliums (HFRPE) expression of major histocompatibility complex (MHC) and costimulatory molecules. Methods. Pure cultures of HFRPE cells were isolated. HFRPE cells were incubated in the presence of Interferon-? (IFN-?), IFN-ß, Tumor Necrosis Factor-a (TNF-a), Interleukin-1ß (IL-1ß), Tumor Growth Factor-ß (TGF-ß), and a combination of IFN-? and TGF-ß (pre-incubation and simultaneously incubated). The expression of MHC class I and class II, Intercellular cell adhesion molecule (ICAM-1), B7-1 (CD80), and B7-2 (CD86) molecules was quantitatively analyzed by flow cytometry. Results. The cultured HFRPE cells expressed high levels of MHC class I and low levels of MHC class II and ICAM-1 molecules. After culture with the above mentioned cytokines, IFN-? up-regulated the HFRPE’s expression of MHC class II and ICAM-1. IFN-ß and IL-1ß only up-regulated the expression of ICAM-1. TGF-ß was unable to suppress the up-regulatory effect of IFN-? in HFRPE cells (pre-incubated and simultaneously incubated). The other cytokines did not have any significant effect on HFRPE’s expression of MHC I and II or the selected costimulatory molecules. Conclusions. Our findings indicate that TGF-ß cannot suppress up-regulating effects of IFN-? on HFRPE’s expression of MHC and costimulatory molecules. Overall, the weak or lack of expression of costimulatory molecules after stimulation with various cytokines further confirms that HFRPE cells are weak antigen presenting cells.

Human fetal retinal pigment epithelium induces apoptosis in human T-cell line Jurkat which is independent from its expression of TRAIL

Purpose. To evaluate whether human fetal retinal pigment epithelial (HFRPE) cells express TRAIL (tumor necrosis factor related apoptosis inducing ligand). The role of TRAIL in HFRPE induced apoptosis was evaluated. Methods. Pure cultures of HFRPE cells were isolated. The expression of TRAIL protein and mRNA in non-activated and IFN-? activated HFRPE cells was evaluated with RT-PCR. The role of TRAIL in HFRPE induced apoptosis was assessed by incubating HFRPE cells with human T-cell leukemia line Jurkat (Jkt) in the presence or absence of neutralizing TRAIL antibodies. Cultures were pulsed with [ 3 H]-thymidine to measure Jkt cell proliferation. The role of TRAIL was further examined by western blott evaluating the cleavage of caspases 8 and 10 in Jkt cells after their incubation with HFRPE cells. Results. HFRPE cells expressed TRAIL mRNA. The expression of TRAIL mRNA and protein was up-regulated by IFN-? activation. However, anti-TRAIL antibodies were not able to prevent the HFRPE induced suppression of Jkt cell proliferation. The caspases 8 and 10 were also not cleaved in Jkt cells after their incubation with IFN-? activated HFRPE cells. Conclusions. Although HFRPE cells express TRAIL and its expression is upregulated by IFN-? activation, TRAIL is not involved in HFRPE induced apoptosis in Jkt cells. Currently the role of TRAIL in HFRPE cells is under investigation.