Mutation of the transcription factor and tumor suppressor gene WT1 results in a range of genitourinary anomalies in humans, including 46,XY gonadal dysgenesis, indicating that WT1 plays a critical role in sex determination. However, because knockout of Wt1 in mice results in apoptosis of the genital ridge, it is unknown whether WT1 is required for testis development after the initial steps of sex determination. To address this question, we generated a mouse strain carrying a Wt1 conditional knockout allele and ablated Wt1 function specifically in Sertoli cells by embryonic day 14.5, several days after testis determination. Wt1 knockout resulted in disruption of developing seminiferous tubules and subsequent progressive loss of Sertoli cells and germ cells such that postnatal mutant testes were almost completely devoid of these cell types and were severely hypoplastic. Thus, Wt1 is essential for the maintenance of Sertoli cells and seminiferous tubules in the developing testes. Of particular note, expression of the testisdetermining gene Sox9 in mutant Sertoli cells was turned off at embryonic day 14.5 after Wt1 ablation, suggesting that WT1 regulates Sox9, either directly or indirectly, after Sry expression ceases. Our data, along with previous work demonstrating the role of Wt1 at early stages of gonadal development, thus indicate that Wt1 is essential at multiple steps in testicular development.Sertoli cell ͉ testicular cord ͉ Mü llerian duct ͉ Amh ͉ Sox8

Significance Patients with compromised T-cell function are at risk for opportunistic fungal infections. We have developed a novel approach to restore immunity by using a fungal pattern-recognition receptor Dectin-1 to redirect T-cell specificity to carbohydrate antigen in the fungal cell wall. We did so by genetically modifying T cells using the nonviral Sleeping Beauty gene-transfer system to enforce expression of a chimeric antigen receptor (CAR) that recapitulates the specificity of Dectin-1 (D-CAR). The D-CAR + T cells can be electroporated and propagated on artificial activating and propagating cells in a manner suitable for human application, enabling this immunology to be translated into immunotherapy. This approach has implications for genetically modifying T cells to express CARs with specificity for carbohydrate and thus broadening their application in the investigational treatment of pathogens and malignancies.

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in >10(9) CAR(+)γδ T cells from <10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.

Renal failure is a frequent and costly complication of many chronic diseases, including diabetes and hypertension. One common feature of renal failure is glomerulosclerosis, the pathobiology of which is unclear. To help elucidate this, we generated a mouse strain carrying the missense mutation Wt1 R394W, which predisposes humans to glomerulosclerosis and early-onset renal failure (Denys-Drash syndrome [DDS]). Kidney development was normal in Wt1؉/R394W heterozygotes. However, by 4 months of age 100% of male heterozygotes displayed proteinuria and glomerulosclerosis characteristic of DDS patients. This phenotype was observed in an MF1 background but not in a mixed B6/129 background, suggestive of the action of a strain-specific modifying gene(s). WT1 encodes a nuclear transcription factor, and the R394W mutation is known to impair this function. Therefore, to investigate the mechanism of Wt1 R394W-induced renal failure, the expression of genes whose deletion leads to glomerulosclerosis (NPHS1, NPHS2, and CD2AP) was quantitated. In mutant kidneys, NPHS1 and NPHS2 were only moderately downregulated (25 to 30%) at birth but not at 2 or 4 months. Expression of CD2AP was not changed at birth but was significantly upregulated at 2 and 4 months. Podocalyxin was downregulated by 20% in newborn kidneys but not in kidneys at later ages. Two other genes implicated in glomerulosclerosis, TGFB1 and IGF1, were upregulated at 2 months and at 2 and 4 months, respectively. It is not clear whether the significant alterations in gene expression are a cause or a consequence of the disease process. However, the data do suggest that Wt1 R394W-induced glomerulosclerosis may be independent of downregulation of the genes for NPHS1, NPHS2, CD2AP, and podocalyxin and may involve other genes yet to be implicated in renal failure. The Wt1 R394W mouse recapitulates the pathology and disease progression observed in patients carrying the same mutation, and the mutation is completely penetrant in male animals. Thus, it will be a powerful and biologically relevant model for investigating the pathobiology of the earliest events in glomerulosclerosis.Glomerulosclerosis, whether primary or secondary to other disease processes, is a key and common feature of progressive renal failure, which is a major cause of morbidity and mortality in the United States. Although several genetic and environmental insults are known to cause primary glomerulosclerosis, the cellular mechanism by which they initiate this process is still largely unknown. A knowledge of these mechanisms would greatly aid in identifying strategies to prevent or slow the development of glomerulosclerosis, regardless of its etiology.Glomeruli are complex and specialized structures responsible for blood filtration in the kidney and are targets of injury in a number of human diseases. The major functional features of the glomerulus are capillary loops lined with fenestrated endothelial cells, supporting mesangial cells, the glomerular basement membrane (GBM), and podocytes. The "octopuslike" ...

One mechanism by which normal cells become converted to tumor cells involves the aberrant transcriptional activation of genes that are normally silent. We characterize a promoter that normally exhibits highly tissue-and stage-specific expression but displays ubiquitous expression when cells become immortalized or malignant, regardless of their lineage or tissue origin. This promoter normally drives the expression of the Pem homeobox gene in specific cell types in ovary and placenta but is aberrantly expressed in lymphomas, neuroblastomas, retinoblastomas, carcinomas, and sarcomas. By deletion analysis we identified a region between nucleotides ؊80 and ؊104 that was absolutely critical for the expression from this distal Pem promoter (Pem Pd). Site-specific mutagenesis and transfection studies revealed that this region contains two consensus Ets sites and a single Sp1 site that were necessary for Pem Pd expression. Gel shift analysis showed that Ets and Sp1 family members bound to these sites. Transfection studies demonstrated that the Ets family members Elf1 and Gabp and the Sp1 family members Sp1 and Sp3 transactivated the Pem Pd. Surprisingly, we found that Sp3 was a more potent activator of the Pem Pd than was Sp1; this is unusual, because Sp3 is either a weak activator or a repressor of most other promoters. Activation by either Elf1 or Gabp required an intact Sp1 family member binding site, suggesting that Ets and Sp1 family members cooperate to activate Pem Pd transcription. Expression from the Pem Pd (either transiently transfected or endogenous) depended on the Ras pathway, which could explain both its Ets-and Sp1-dependent expression in normal cells and its aberrant expression in tumor cells, in which ras protooncogenes are frequently mutated. We suggest that the Pem Pd may be a useful model system to understand the molecular mechanism by which a tissue-specific promoter can be corrupted in tumor cells.