Martha B. Reich

Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells

B lymphocytes that recognize soluble self-Ags are routinely found in normal individuals in a functionally inactive or anergic state. Current models indicate that this tolerant state is maintained by interactions with self-Ags that uncouple the BCR from downstream signaling pathways and increase levels of free calcium. Contrary to this expectation, B cells that harbor anti-insulin Ig transgenes (125Tg) are maintained in a tolerant state even though free calcium levels remain normal and tyrosine kinase substrate phosphorylation is preserved following BCR stimulation. Under basal conditions, intracellular levels of inositol 1,4,5-trisphosphate are increased and NFATc1 levels are reduced in 125Tg B cells. The 125Tg B cells are markedly impaired in their ability to mobilize calcium upon stimulation with ionomycin, and BCR-induced calcium mobilization from internal stores is decreased. In contrast, poisoning intracellular calcium pumps with thapsigargin increases calcium mobilization in 125Tg B cells. Changes in calcium signaling are accompanied by a failure of 125Tg B cells to translocate NFATc1 into the nucleus following stimulation with either anti-IgM or ionomycin. Thus, disassociation of BCR from multiple signaling pathways is not essential for maintaining tolerance in anti-insulin 125Tg B cells. Rather, BCRs that are occupied by autologous insulin deliver signals that induce changes in intracellular calcium mobilization and maintain tolerance by preventing activation of key transcription factors such as NFAT.

Fibroblast growth factor receptor-1 interacts with the T-cell receptor signalling pathway

Fibroblast growth factor receptors are expressed by some T cells, and provide costimulation for these cells. Such receptors allow T cells to respond to fibroblast growth factors expressed in response to injury and inflammation and may provide a mechanism for ‘context‐dependent’ responses to antigens within the local microenvironment. The mechanisms by which fibroblast growth factor receptors might interact with the TCR signalling pathway are not defined. Here we show that the TCR and fibroblast growth factor receptors co‐localize during combined stimulation. Signalling via fibroblast growth factor receptors alone results in phosphorylation of Lck and induces nuclear translocation of nuclear factors of activated T cells. Combined stimulation via fibroblast growth factor receptors and the TCR synergistically enhances the activation of nuclear factors of activated T cells. The results suggest that peptide growth factors produced at sites of injury and inflammation can contribute to the outcome of T‐cell encounters with antigen.

Autoreactive T cells from a type I diabetic recognize multiple class II products

Mononuclear cells obtained from a child at the acute presentation of type I diabetes were stimulated in vitro with human insulin followed by IL-2 and IL-4. All of the T-cell clones isolated from this stimulation were autoreactive, recognizing autologous B cells in the absence of insulin or other exogenous antigens. Eleven CD4+ clones were studied in detail to identify the class II MHC antigens stimulating these autoreactive cells. The donor was heterozygous for DR3DQw2 and DR4DQw3.2 haplotypes, a combination of alleles with a greatly increased risk for type I diabetes. The clones demonstrated skewed recognition of class II antigens. Three clones appeared to recognize a peptide derived from one class II beta chain (DR beta 1, DR4Dw4) presented by another class II beta chain (DR beta 4, DRw53). Three clones were stimulated by cells expressing DPw4 molecules. Only one clone recognized a product derived from the DR3 haplotype. In contrast to both antigen-specific and autoreactive T-cell clones derived from normal individuals, many of the autoreactive T cells isolated from this subject were stimulated by class II molecules other than DR beta 1. The results support the hypothesis that autoreactive T cells recognize autologous peptides in association with MHC and some of these peptides are derived from self MHC molecules.

Inhibition of Src kinases combined with CD40 ligand blockade prolongs murine cardiac allograft survival

Background. Members of the Src family of tyrosine kinases (SFKs) are requisite signaling molecules activated by multiple receptors during immune responses. Their expression and catalytic activity has not been characterized in allograft rejection in vivo. Methods. We measured expression and catalytic activity of SFKs in MHC- mismatched murine cardiac allografts. We also examined the effects of a Src inhibitor (CGP77675) with or without anti-CD154 mAb on graft survival, histology, and expression and catalytic activity of SFKs within the grafts. Results. In acutely rejecting allografts from untreated controls, total activity of Hck and Lyn increased 10-fold, predominantly reflecting increases in the amount of protein. Total activity of Lck increased only fourfold, reflecting small changes in both the amount of protein and specific activity. One dose of anti-CD154 plus CGP77675 markedly diminished cellular infiltration, but survival was only moderately prolonged despite inhibition of all SFKs in the rejected grafts. Two doses of anti-CD154 plus CGP77675 allowed permanent graft acceptance in 60% of recipients even after discontinuation of the inhibitor. Both rejected and long surviving grafts showed increased activity of all SFKs. Recipients that rejected their grafts showed serum alloantibody production, and grafts rejected during treatment demonstrated deposition of complement indicating the contribution of antibody to rejection. Conclusions. The myeloid and B cell Src family kinases, Hck and Lyn, rather than the T cell Src kinase Lck, show the greatest increase in expression and total activity in rejecting allografts. Both rejected and long-surviving grafts show significant increases in SFK expression and acitivity.