Changes in autoreactive T cell avidity during type 1 diabetes development.

The activation threshold for antigen-specific T cell responses is dependent on the avidity of the trimolecular interaction between TCR, antigen, and MHC. We compared CD4+ T cell avidities for the diabetes-associated autoantigen glutamic acid decarboxylase 555-567 (GAD 555) among serial samples from autoantibody-positive subjects at high risk of progression to type 1 diabetes (T1D). T cells from three at-risk subjects demonstrated significant avidity increases (p<0.05 by F test) over time. This avidity shift correlated with the outgrowth of T cells expressing TCR BV 9, 15, 17 or 20 that demonstrated higher GAD 555 tetramer-binding levels compared to cells expressing other TCR BV genes. Similar analysis of autoantibody-negative, first-degree relatives and T1D patients did not demonstrate similar changes in avidity. These data implicate the outgrowth of T cells expressing higher affinity TCR in a process of antigen-specific T cell avidity maturation during the pre-clinical stage of T1D.

The antiapoptotic gene A1/BFL1 is a WT1 target gene that mediates granulocytic differentiation and resistance to chemotherapy.

Previous work has demonstrated that WT1 (-Ex5/-KTS) potentiates granulocyte colony-stimulating factor (G-CSF)-mediated granulocytic differentiation. This WT1 isoform suppresses cyclin E, which may contribute to the prodifferentiation effect by slowing proliferation, but WT1 target genes that affect survival might also be involved. We screened a cDNA array and identified the bCL2 family member A1/BFL1 as a new WT1 target gene in 32D cl3 murine myeloblast cells. Induction of WT1 (-Ex5/-KTS) expression is accompanied by up-regulation of A1 on the cDNA array, and this up-regulation was confirmed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Moreover, both promoter-reporter assays and chromatin immunoprecipitation assays suggest that this isoform of WT1 activates the promoter directly. Constitutive expression of A1 in 32D cl3 cells induces spontaneous granulocytic differentiation, with both morphologic and cell-surface antigen changes, as well as resistance both to chemotherapy and to withdrawal of interleukin-3 (IL-3). Finally, we note an association between WT1 expression and A1 expression in primary acute myeloid leukemia samples. Taken together, these results demonstrate that A1 is a new WT1 target gene involved in both granulocytic differentiation and resistance to cell death, and suggests that these genes might play an important role in the biology of high-risk leukemias.

Cyclin E is a target of WT1 transcriptional repression.

WT1 was originally identified as a Wilms' tumor suppressor gene, but it may have oncogenic potential in leukemia and in some solid tumors. WT1 is a transcription factor that has been implicated in the regulation of target genes related to apoptosis, genitourinary differentiation, and cell cycle progression. Because induction of WT1 leads indirectly to increased p21 expression in osteosarcoma cells, we investigated the possibility that other genes involved in the G(1)/S phase transition might also be WT1 targets. Cyclin E plays a crucial role in the cell cycle by activating cyclin-dependent kinase 2, which phosphorylates Rb, leading to progression from G(1) into S phase. We identified several WT1 binding sites in the cyclin E promoter. We demonstrate that WT1 binds to these sites and that in transient transfection assays WT1 represses the cyclin E promoter. This activity is dependent on the presence of a binding site located downstream of the transcription start site. In intact cells, induction of WT1 expression down-regulates cyclin E protein levels. These results provide the first demonstration that WT1 can directly modulate the expression of a gene involved in cell cycle progression.