No abstract

Insulin-dependent diabetes mellitus (IDDM) is a disease with an autoimmune aetiology. The inbred non-obese diabetic (NOD) mouse strain provides a good animal model of the human disease and genetic analysis suggests that, as in man, at least one of the several genes controlling the development of IDDM is linked to the major histocompatibility complex. The NOD mouse does not express I-E owing to a deletion in the promoter region of the I-E alpha-chain gene, and the sequence of NOD I-A beta-chain in the first external domain is unique with His 56 and Ser 57 replacing Pro and Asp, respectively, at these positions. There has been considerable interest in the role amino acid 57 might have in conferring susceptibility to autoimmune diseases, including IDDM. The presence of a charged residue (such as Asp) at this position might affect the conformation of the peptide binding groove. But it could be assumed that Pro 56 gives rise to a different conformation of I-A beta-chain than does His 56. We therefore constructed transgenic NOD mice in which the transgene encoded a modified A beta nod with Pro 56, and studied its effect on the development of IDDM in this mouse strain. Previous studies have suggested that NOD mice expressing I-E as a result of the introduction of an I-E alpha-chain (E alpha) transgene are protected from the development of insulitis and hence IDDM. To explore further the protective effect of this molecule we constructed a second class of transgenic NOD mouse carrying an E alpha d transgene. Both transgenes protected the mice from IDDM, but this was not associated with a complete deletion of any T cells expressing commonly used T-cell receptor V beta genes.

Most tumour cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and evade apoptosis. Intriguingly, the molecular mechanisms that link the Warburg effect with the suppression of apoptosis are not well understood. In this study, using loss-of-function studies in vitro and in vivo, we show that the anti-apoptotic protein poly(ADP-ribose) polymerase (PARP)14 promotes aerobic glycolysis in human hepatocellular carcinoma (HCC) by maintaining low activity of the pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. Notably, PARP14 is highly expressed in HCC primary tumours and associated with poor patient prognosis. Mechanistically, PARP14 inhibits the pro-apoptotic kinase JNK1, which results in the activation of PKM2 through phosphorylation of Thr365. Moreover, targeting PARP14 enhances the sensitization of HCC cells to anti-HCC agents. Our findings indicate that the PARP14-JNK1-PKM2 regulatory axis is an important determinant for the Warburg effect in tumour cells and provide a mechanistic link between apoptosis and metabolism.

TCR signal strength is involved in many cell fate decisions in the T-cell lineage. Here, we show that transcriptional events induced by Hedgehog (Hh) signaling reduced TCR signal strength in mice. Activation of Hh signaling in thymocytes in vivo by expression of a transgenic transcriptional-activator form of Gli2 (Gli2ΔN2) changed the outcome of TCR ligation at many stages of thymocyte development, allowing self-reactive cells to escape clonal deletion; reducing transgenic TCR-mediated positive selection; reducing the ratio of CD4/CD8 single-positive (SP) cells; and reducing cell surface CD5 expression. In contrast, in the Shh−/− thymus the ratio of CD4/CD8 cells and both positive and negative selection of a transgenic TCR were increased, demonstrating that Shh does indeed influence TCR repertoire selection and the transition from double-positive (DP) to SP cell in a physiological situation. In peripheral T cells, Gli2ΔN2 expression attenuated T-cell activation and proliferation, by a mechanism upstream of ERK phosphorylation.

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

SummaryConstitutive NF-κB signaling promotes survival in multiple myeloma (MM) and other cancers; however, current NF-κB-targeting strategies lack cancer cell specificity. Here, we identify the interaction between the NF-κB-regulated antiapoptotic factor GADD45β and the JNK kinase MKK7 as a therapeutic target in MM. Using a drug-discovery strategy, we developed DTP3, a D-tripeptide, which disrupts the GADD45β/MKK7 complex, kills MM cells effectively, and, importantly, lacks toxicity to normal cells. DTP3 has similar anticancer potency to the clinical standard, bortezomib, but more than 100-fold higher cancer cell specificity in vitro. Notably, DTP3 ablates myeloma xenografts in mice with no apparent side effects at the effective doses. Hence, cancer-selective targeting of the NF-κB pathway is possible and, at least for myeloma patients, promises a profound benefit.

Although the underlying mechanisms are not well understood, it is generally believed that antigen recognition by T cells in the absence of costimulation may alter the immune response, leading to anergy or tolerance. Further support for this concept comes from animal models of autoimmunity and transplantation, where treatments based on costimulation blockade, in particular CD40 ligand (CD40L)-specific antibodies, have been highly effective. We investigated the mechanisms of action of an antibody to CD40L and provide evidence that its effects are dependent on the constant (Fc) region. Prolongation of graft survival is dependent on both complement- and Fc receptor-mediated mechanisms in a major histocompatibility complex (MHC)-mismatched skin transplant model. These data suggest that antibodies to CD40L act through selective depletion of activated T cells, rather than exerting immune modulation by costimulation blockade as currently postulated. This finding opens new avenues for treatment of immune disorders based on selective targeting of activated T cells.

In vitro, engagement of GITR on Treg cells by the agonistic anti-GITR mAb, DTA-1, appears to abrogate their suppressive function. The consequence of in vivo engagement of GITR by DTA-1 is, however, less clear. In this study, we show that Treg cells isolated from DTA-1-treated mice were as potent as those from untreated mice in suppressing conventional CD4 T cells in vitro, indicating that in vivo GITR ligation does not disable Treg cells. Treatment of Foxp3/GFP knock-in mice with DTA-1 led to a selective reduction of circulating Treg cells, suggesting that DTA-1 is a depleting mAb which preferentially targets Treg cells. In tumour-bearing mice, DTA-1-mediated depletion of Treg cells was most marked in tumours but not in tumour-draining lymph node. These features were confirmed in an adoptive transfer model using tumour antigen-specific Treg cells. Interestingly, Treg cells detected in tumour tissues expressed much higher levels of GITR than those in tumour-draining lymph nodes, indicating that the efficiency of depletion might be correlated with the level of GITR expression. Finally, in vivo labelling of GITR in naive or tumour-bearing mice demonstrated that Treg cells constitutively expressed higher levels of GITR than conventional T cells, independent of location and activation state, consistent with the preferential in vivo depletion of Tregs by DTA-1. Thus, depletion of Treg cells represents a previously unrecognised in vivo activity of DTA-1 which has important implications for the application of anti-GITR antibodies in cancer immunotherapy.