John Colgan

TCR stimulation with modified anti-CD3 mAb expands CD8+ T cell population and induces CD8+CD25+ Tregs.

Modified anti-CD3 mAbs are emerging as a possible means of inducing immunologic tolerance in settings including transplantation and autoimmunity such as in type 1 diabetes. In a trial of a modified anti-CD3 mAb [hOKT3gamma1(Ala-Ala)] in patients with type 1 diabetes, we identified clinical responders by an increase in the number of peripheral blood CD8+ cells following treatment with the mAb. Here we show that the anti-CD3 mAb caused activation of CD8+ T cells that was similar in vitro and in vivo and induced regulatory CD8+CD25+ T cells. These cells inhibited the responses of CD4+ cells to the mAb itself and to antigen. The regulatory CD8+CD25+ cells were CTLA4 and Foxp3 and required contact for inhibition. Foxp3 was also induced on CD8+ T cells in patients during mAb treatment, which suggests a potential mechanism of the anti-CD3 mAb immune modulatory effects involving induction of a subset of regulatory CD8+ T cells.

Functional interaction between p53, the TATA-binding protein (TBP), andTBP-associated factors in vivo.

The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAFII230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAFII40 and TAFII60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAFII110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53.

Blockade of Late Stages of Autoimmune Diabetes by Inhibition of the Receptor for Advanced Glycation End Products

Ligation of the receptor for advanced glycation end products (RAGE) occurs during inflammation. Engagement of RAGE results in enhanced expression of addressins and it is therefore, not surprising that previous studies have shown a role of RAGE/ligand interactions in immune responses including cell/cell contact but the role of RAGE in spontaneous autoimmunity has not been clearly defined. To study the role of RAGE/ligand interactions in autoimmune diabetes, we tested the ability of soluble RAGE, a scavenger of RAGE ligands, in late stages of diabetes development in the NOD mouse-disease transferred with diabetogenic T cells and recurrent disease in NOD/scid recipients of syngeneic islet transplants. RAGE expression was detected on CD4+, CD8+, and B cells from diabetic mice and transferred to NOD/scid recipients. RAGE and its ligand, S100B, were found in the islets of NOD/scid mice that developed diabetes. Treatment of recipient NOD/scid mice with soluble RAGE prevented transfer of diabetes and delayed recurrent disease in syngeneic islet transplants. RAGE blockade was associated with increased expression of IL-10 and TGF-β in the islets from protected mice. RAGE blockade reduced the transfer of disease with enriched T cells, but had no effect when diabetes was transferred with the activated CD4+ T cell clone, BDC2.5. We conclude that RAGE/ligand interactions are involved in the differentiation of T cells to a mature pathogenic phenotype during the late stages of the development of diabetes.

Cyclophilin A-Deficient Mice Are Resistant to Immunosuppression by Cyclosporine

Cyclosporine is an immunosuppressive drug that is widely used to prevent organ transplant rejection. Known intracellular ligands for cyclosporine include the cyclophilins, a large family of phylogenetically conserved proteins that potentially regulate protein folding in cells. Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that binds to and inhibits calcineurin, a serine/threonine phosphatase that is activated by TCR engagement. Amino acids within the cyclophilins that are critical for binding to cyclosporine have been identified. Most of these residues are highly conserved within the 15 mammalian cyclophilins, suggesting that many are potential targets for the drug. We examined the effects of cyclosporine on immune cells and mice lacking Ppia, the gene encoding the prototypical cyclophilin protein cyclophilin A. TCR-induced proliferation and signal transduction by Ppia−/− CD4+ T cells were resistant to cyclosporine, an effect that was attributable to diminished calcineurin inhibition. Immunosuppressive doses of cyclosporine failed to block the responses of Ppia−/− mice to allogeneic challenge. Rag2−/− mice reconstituted with Ppia−/− splenocytes were also cyclosporine resistant, indicating that this property is intrinsic to Ppia−/− immune cells. Thus, among multiple potential ligands, CypA is the primary mediator of immunosuppression by cyclosporine.

A direct interaction between a glutamine-rich activator and the N terminus of TFIIB can mediate transcriptional activation in vivo.

Studies examining the mechanism by which transcriptional activators function have suggested that the general transcription factor IIB (TFIIB) can be a target for certain regulatory proteins. For example, we showed previously that expression of a mutant form of TFIIB can specifically inhibit activation in vivo mediated by the strong, glutamine-rich activator protein GAL4-ftzQ. Using transient cotransfection assays, we have defined the regions in both GAL4-ftzQ and TFIIB that are required for activity in vivo and provide evidence that a potential zinc finger structure at the N terminus of TFIIB is necessary for the observed functional interaction between the two proteins. Using a protein binding assay, we have demonstrated that GAL4-ftzQ can specifically interact with TFIIB in vitro. This interaction requires the same regions in both molecules necessary for function in vivo and is reduced or eliminated by mutations predicted to disrupt the zinc finger in TFIIB. These results support the idea that a direct interaction between a regulatory protein and TFIIB can be important for transcriptional activation in vivo and, combined with previous data of others, suggest that different activators can function by contacting distinct regions of TFIIB.