5T4 oncofetal antigen is expressed in high risk of relapse childhood pre-B acute lymphoblastic leukemia and is associated with a more invasive and chemotactic phenotype.

Although the overall prognosis in childhood acute lymphoblastic leukemia (ALL) is good, outcome after relapse is poor. Recurrence is frequently characterized by the occurrence of disease at extramedullary sites, such as the central nervous system and testes. Subpopulations of blasts able to migrate to such areas may have a survival advantage and give rise to disease recurrence. Gene expression profiling of 85 diagnostic pre-B-ALL bone marrow samples revealed higher 5T4 oncofetal antigen transcript levels in cytogenetic high-risk subgroups of patients (P<0.001). Flow cytometric analysis determined that bone marrow from relapse patients have a significantly higher percentage of 5T4-positive leukemic blasts than healthy donors (P=0.005). The high-risk Sup-B15 pre-B-ALL line showed heterogeneity in 5T4 expression, and the derived, 5T4(+) (Sup5T4) and 5T4(-) (Sup) subline cells, displayed differential spread to the omentum and ovaries following intraperitoneal inoculation of immunocompromised mice. Consistent with this, Sup5T4 compared with Sup cells show increased invasion in vitro concordant with increased LFA-1 and VLA-4 integrin expression, adhesion to extracellular matrix and secretion of matrix metalloproteases (MMP-2/-9). We also show that 5T4-positive Sup-B15 cells are susceptible to 5T4-specific superantigen antibody-dependent cellular toxicity providing support for targeted immunotherapy in high-risk pre-B-ALL.

Novel IFNγ ELISPOT assay for detection of functional carcinoembryonic antigen-specific chimeric antigen receptor-redirected T cells.

We here describe the development of a novel ELISPOT assay for the detection and enumeration of IFNγ-secreting functional chimeric antigen receptor (CAR)-redirected T cells against carcinoembryonic antigen (CEA). This method is valuable for clinical trials to monitor the presence of functional CEA-specific T cells transduced with a CAR. The same principle should be applicable for the detection of functional CAR-redirected T cells against any other tumour-associated antigens by immobilizing a particular biotinylated antigen to streptavidin-coated beads.

Monocyte chemoattractant protein-1 has prosclerotic effects both in a mouse model of experimental diabetes and in vitro in human mesangial cells.

AIMS/HYPOTHESIS: Diabetic nephropathy is characterised by mesangial extracellular matrix accumulation. Monocyte chemoattractant protein-1 (MCP-1), a chemokine promoting monocyte infiltration, is upregulated in the diabetic glomerulus. We performed in vitro and in vivo studies to examine whether MCP-1 may have prosclerotic actions in the setting of diabetes, presumably via its receptor, chemokine (C-C motif) receptor 2 (CCR2), which has been described in mesangial cells. METHODS: Human mesangial cells were exposed to recombinant human (rh)-MCP-1 (100 ng/ml) for 12, 24 and 48 h and to rh-MCP-1 (10, 100 and 200 ng/ml) for 24 h. Fibronectin, collagen IV and transforming growth factor, beta 1 (TGF-beta1) protein levels were measured by ELISA and pericellular polymeric fibronectin levels by western blotting. The intracellular mechanisms were investigated using specific inhibitors for CCR2, nuclear factor kappa B (NF-kappaB), p38 mitogen-activated protein kinase and protein kinase C, and an anti-TGF-beta1 blocking antibody. In both non-diabetic and streptozotocin-induced diabetic mice that were deficient or not in MCP-1, glomerular fibronectin accumulation was examined by immunohistochemistry, while cortical Tgf-beta1 (also known as Tgfb1) and fibronectin mRNA and protein levels were examined by real-time PCR and western blotting. RESULTS: In mesangial cells, MCP-1 binding to CCR2 induced a 2.5-fold increase in fibronectin protein levels at 24 h followed by a rise in pericellular fibronectin, whereas no changes were seen in collagen IV production. MCP-1-induced fibronectin production was TGF-beta1- and NF-kappaB-dependent. In diabetic mice, loss of MCP-1 diminished glomerular fibronectin protein production and both renal cortical Tgf-beta1 and fibronectin mRNA and protein levels. CONCLUSIONS/INTERPRETATION: Our in vitro and in vivo findings indicate a role for the MCP-1/CCR2 system in fibronectin deposition in the diabetic glomerulus, providing a new therapeutic target for diabetic nephropathy.

Effect of TA-CIN (HPV 16 L2E6E7) booster immunisation in vulval intraepithelial neoplasia patients previously vaccinated with TA-HPV (vaccinia virus encoding HPV 16/18 E6E7).

Heterologous prime-boost vaccination schedules employing TA-HPV, a vaccinia virus encoding HPV 16/18 E6 and E7, in combination with TA-CIN, an HPV 16 L2E6E7 fusion protein, may offer advantages over the use of either agent alone for the immunotherapy of human papillomavirus (HPV) type 16-associated vulval intraepithelial neoplasia (VIN). In the present study, 10 women with HPV 16-positive high grade VIN, previously primed with TA-HPV, received three booster immunisations with TA-CIN. All but one demonstrated HPV 16-specific proliferative T-cell and/or serological responses following vaccination. Three patients additionally showed lesion shrinkage or symptom relief, but no direct correlation between clinical and immunological responses was seen.

P38 mitogen-activated protein kinase mediates hexosamine-induced TGFbeta1 mRNA expression in human mesangial cells.

AIMS/HYPOTHESIS: The hexosamine pathway has been implicated in the induction of TGFbeta1 expression and in the pathophysiology of diabetic glomerulopathy. Glucose-induced TGFbeta1 expression is mediated by p38 mitogen-activated-protein-kinase (p38-MAPK) and this kinase is activated in the diabetic glomeruli. We examined whether the p38-MAPK is implicated in hexosamine-induced TGFbeta1 mRNA expression in human mesangial cells. GFAT overexpression induced an increase in p38-MAPK activation after 6 and 12 h incubation in normal glucose, and this was prevented by the GFAT inhibitor azaserine. Furthermore, high glucose enhanced p38-MAPK activation in GFAT tranfected cells ( p

Human T cell responses to HPV 16 E2 generated with monocyte-derived dendritic cells.

Persistent infection with human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical cancer. The E2 protein is required early in viral infection and therefore may serve as a useful immune target for a vaccine aimed at prevention or therapy of premalignant lesions. Dendritic cells (DC) prepared from monocytes and pulsed with bacterially produced HPV 16 E2 C-terminus protein were used to stimulate autologous T cells over several rounds of stimulation. T cells were tested for gamma-interferon release by ELISPOT and for cytotoxic activity by (51)chromium release assays. To generate E2-expressing target cells for cytotoxicity assays, we constructed a recombinant vaccinia virus encoding HPV 16 E2, which was used to infect autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL). The results show that DC pulsed with E2 C-terminus protein induce gamma-interferon-releasing T cells as demonstrated by ELISPOT. Furthermore, we demonstrate E2-specific lysis of vaccinia-E2 infected autologous LCL by CD8+ cytotoxic T lymphocytes (CTL). E2-specific CTL did not lyse untreated autologous LCL or LCL infected with wild-type vaccinia and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. In addition, T cells stimulated with DC in the absence of E2 failed to demonstrate lysis of vaccinia-E2-labeled targets. Phenotypically, CTL populations were CD3+/CD8+. These results will facilitate the study of naturally occurring T-cell responses to HPV E2 in patients with cervical intraepithelial neoplasia and the development of immunotherapeutic strategies designed to treat this and other HPV-associated diseases.

Multiple mechanisms underlie HLA dysregulation in cervical cancer.

The consistent dysregulation of HLA expression in cervical neoplasia is likely to influence the natural history of the disease and prospects for cell-mediated vaccine therapies. We have studied the underlying mechanisms in eight new cervical cancer cell lines derived from primary tumour biopsies. At least five independent mechanisms leading to changes in HLA expression were seen: HLA class I allelic transcription but no protein; abnormal HLA class I allelic transcription; no HLA-B locus transcription; loss of heterozygosity (LOH); no gammaIFN-mediated upregulation of HLA class I expression, and/or no interferon-gamma (gammaIFN)-mediated HLA class II induction. These were evident in different combinations in 7/8 cell lines showing that multiple, mostly irreversible mechanisms not overridden by gammaIFN, are responsible for HLA dysregulation in cervical neoplasia. Point mutations were responsible for lack of HLA-A2 expression in two cases. In cell line 808, the mutation encodes a stop codon in exon 3; in cell line 778, mutation of the first intron acceptor site leads to use of an alternative AG site in exon 2, resulting in a frameshift and a stop codon after the translation of only 38 amino acids. Tumour cells showing specific HLA class I loss may have selective advantage in the face of tumour-specific cytotoxic T cells (CTL). Such immune escape mechanisms present a major obstacle for the success of CTL-mediated therapies in cervical cancer.

Integration of high-risk human papillomavirus DNA is linked to the down-regulation of class I human leukocyte antigens by steroid hormones in cervical tumor cells.

A crucial event in the malignant progression of cervical intraepithelial neoplasia appears to be the up-regulation of high-risk human papillomavirus (HPV) early gene expression. Steroid hormones have been linked to the progression from premalignant to neoplastic status in HPV positive lesions. This report demonstrates that at physiological levels, the glucocorticoid hormone hydrocortisone consistently down-regulates class I human leukocyte antigen (HLA) surface expression in HPV-positive cervical tumor cells but can up-regulate expression in HPV-negative epithelial tumor lines. Suppression of HLA expression was also seen with progesterone, another steroid hormone. The hydrocortisone-mediated modulation of HLA expression is dependent on integration and transcription of the HPV genome and can be blocked by Ru38486, an antagonist of both glucocorticoid and progesterone receptors, indicating the role of these receptors in mediating HLA suppression. The data suggest that HPV integration events in cervical epithelia correlate with hormone-dependent HLA suppression, possibly contributing to the avoidance of tumor recognition by cytotoxic T cells. These studies imply that clinical use of steroids may be contraindicated in HPV-positive individuals who have early premalignant cervical disease or neoplasia but provide evidence that the antiprogestin Ru38486 may be useful in the management of early stage cervical disease.

Identification of a naturally processed HLA A0201-restricted viral peptide from cells expressing human papillomavirus type 16 E6 oncoprotein.

Human papillomavirus (HPV) DNA encoding the oncogenic proteins E6 and E7 is usually retained in cervical carcinomas, implicating these proteins as potential target antigens for immune recognition in this virally associated tumor. We have characterized endogenously processed peptides eluted from major histocompatibility complex class I molecules in cells infected with a recombinant vaccinia expressing the HPV-16 E6 oncoprotein. The reverse-phase chromatography profile of peptides eluted from isolated HLA-A0201 molecules in cells expressing the E6 oncoprotein differs from that of cells not expressing E6. Sequential Edman degradation of novel peaks found in the peptide profiles from cells expressing HPV-16 E6 led to the identification of a naturally processed HLA-A0201-restricted E6 peptide of sequence KLPQLCTEL. This approach has allowed the identification of a viral peptide which is processed and presented by cells expressing the E6 oncoprotein and is a likely target for cytotoxic T lymphocyte recognition in HLA-A0201-positive patients.