Jean-Hubert Caberg

The cross-talk between dendritic and regulatory T cells: good or evil?

Immune responses against pathogens require fine regulation to avoid excessive inflammation, which could be harmful to the host. Moreover, the immune system must be tolerant to nonpathogenic antigens to prevent allergy, autoimmunity, and transplant rejection. There is accumulating evidence that interactions between dendritic cells (DC) and regulatory T (Treg) cells play a crucial role in the balance between immune response and tolerance. Communications between these cells are complex, bidirectional, and mediated by soluble or cell surface molecules. The maturation status of DC, which may be influenced by different microenvironmental factors, is considered as an important checkpoint for the induction of peripheral tolerance through modifications of the activation status of T cells. Moreover, several lines of experimental evidence suggest that different subsets or the functional status of DC are also involved in the promotion of Treg cell differentiation. A better knowledge of the regulatory mechanisms of the immune response induced or inhibited by DC via their interactions with Treg cells could be relevant for the development of new, immunotherapeutic approaches.

E-cadherin-dependent adhesion of dendritic and Langerhans cells to keratinocytes is defective in cervical human papillomavirus-associated (pre)neoplastic lesions

Although human papillomavirus (HPV) DNA is detected in the majority of squamous intraepithelial lesions (SILs) and squamous cell carcinomas (SCCs) of the uterine cervix, the persistence and progression of cervical lesions suggest that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most SILs show quantitative and functional alterations of Langerhans cells (LCs). The aim of this study was to determine whether modulation of E‐cadherin‐mediated homophilic and heterotypic interactions between keratinocytes and LCs is involved in these abnormalities of LCs in (pre)neoplastic cervical epithelium. Cell membrane expression of E‐cadherin and the density of CD1a+ LCs were low in the epithelium of SILs and SCC biopsy specimens, compared with normal exocervical epithelium. Dendritic cells (DCs) and LCs generated in vitro were randomly distributed throughout the full thickness of organotypic cultures of E‐cadherin− HPV‐transformed cells. In contrast, these cells rapidly adhered to the keratinocyte cell layers when HPV‐transformed cells transfected with E‐cadherin were used. These data suggest that the E‐cadherin‐mediated contact between keratinocytes and LCs is potentially important for initiating or maintaining the immune response during chronic HPV infection. Copyright

Silencing of E7 oncogene restores functional E-cadherin expression in human papillomavirus 16-transformed keratinocytes

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intra-epithelial lesions show quantitative and functional alterations of Langerhans cells (LCs). Moreover, E-cadherin-dependent adhesion of LC to keratinocytes (KCs) is defective in cervical HPV16-associated (pre)neoplastic lesions. The possible role of viral oncoprotein E7 in the reduced levels of cell surface E-cadherin was investigated by silencing HPV16 E7 by RNA interference (siRNA). This treatment induced an increased cell surface E-cadherin expression in HPV16-positive KC and a significant adhesion of LC to these squamous cells. The E-cadherin re-expression following HPV16 E7 silencing was associated with increased detection levels of retinoblastoma protein and the activating protein (AP)-2alpha transcription factor. These data suggest that HPV16 E7-induced alterations of LC/KC adhesion may play a role in the defective immune response during cervical carcinogenesis.

X-linked acrogigantism syndrome: clinical profile and therapeutic responses.

X-linked acrogigantism (X-LAG) is a new syndrome of pituitary gigantism, caused by microduplications on chromosome Xq26.3, encompassing the gene GPR101, which is highly upregulated in pituitary tumors. We conducted this study to explore the clinical, radiological, and hormonal phenotype and responses to therapy in patients with X-LAG syndrome. The study included 18 patients (13 sporadic) with X-LAG and microduplication of chromosome Xq26.3. All sporadic cases had unique duplications and the inheritance pattern in two families was dominant, with all Xq26.3 duplication carriers being affected. Patients began to grow rapidly as early as 2-3 months of age (median 12 months). At diagnosis (median delay 27 months), patients had a median height and weight standard deviation scores (SDS) of >+3.9 SDS. Apart from the increased overall body size, the children had acromegalic symptoms including acral enlargement and facial coarsening. More than a third of cases had increased appetite. Patients had marked hypersecretion of GH/IGF1 and usually prolactin, due to a pituitary macroadenoma or hyperplasia. Primary neurosurgical control was achieved with extensive anterior pituitary resection, but postoperative hypopituitarism was frequent. Control with somatostatin analogs was not readily achieved despite moderate to high levels of expression of somatostatin receptor subtype-2 in tumor tissue. Postoperative use of adjuvant pegvisomant resulted in control of IGF1 in all five cases where it was employed. X-LAG is a new infant-onset gigantism syndrome that has a severe clinical phenotype leading to challenging disease management.

Clinical and genetic characterization of pituitary gigantism: an international collaborative study in 208 patients

Despite being a classical growth disorder, pituitary gigantism has not been studied previously in a standardized way. We performed a retrospective, multicenter, international study to characterize a large series of pituitary gigantism patients. We included 208 patients (163 males; 78.4%) with growth hormone excess and a current/previous abnormal growth velocity for age or final height >2 s.d. above country normal means. The median onset of rapid growth was 13 years and occurred significantly earlier in females than in males; pituitary adenomas were diagnosed earlier in females than males (15.8 vs 21.5 years respectively). Adenomas were ≥10 mm (i.e., macroadenomas) in 84%, of which extrasellar extension occurred in 77% and invasion in 54%. GH/IGF1 control was achieved in 39% during long-term follow-up. Final height was greater in younger onset patients, with larger tumors and higher GH levels. Later disease control was associated with a greater difference from mid-parental height (r=0.23, P=0.02). AIP mutations occurred in 29%; microduplication at Xq26.3 - X-linked acrogigantism (X-LAG) - occurred in two familial isolated pituitary adenoma kindreds and in ten sporadic patients. Tumor size was not different in X-LAG, AIP mutated and genetically negative patient groups. AIP-mutated and X-LAG patients were significantly younger at onset and diagnosis, but disease control was worse in genetically negative cases. Pituitary gigantism patients are characterized by male predominance and large tumors that are difficult to control. Treatment delay increases final height and symptom burden. AIP mutations and X-LAG explain many cases, but no genetic etiology is seen in >50% of cases.

Defensins induce the recruitment of dendritic cells in cervical human papillomavirus-associated (pre)neoplastic lesions formed in vitro and transplanted in vivo

In addition to their direct antimicrobial activity, defensins might also influence adaptive immu‐nity by attracting immature dendritic cells (DC). As these cells have been shown to be deficient in uterine cervix carcinogenesis, we evaluated the ability of a‐de‐fensin (HNP‐2, human neutrophil defensin 2) and ß‐defensin (HßD2, human beta defensin 2) to stimulate their migration in human papillomavirus (HPV)‐associated (pre)cancers. We first observed, using RT‐PCR and immunohistology, that HßD2 is absent in HPV‐transformed keratinocytes and that it is weakly expressed in cervical (pre)neoplastic lesions in comparison with normal keratinocytes. We next demonstrated that defensins exert a chemotactic activity for DC in a Boyden Chamber assay and stimulate their infiltration in an in vitro‐formed (pre)neoplastic epithelium (orga‐notypic culture of HPV‐transformed keratinocytes). To evaluate the ability of defensins also to recruit DC in vivo, we developed a model of immunodeficient mice grafted with organotypic cultures of HPV+ keratino‐cytes, which form an epithelium similar to a high‐grade neoplastic lesion, with tumoral invasion and neovascu‐larization. Intravenously injected human DC were able to infiltrate grafts of HPV+ keratinocytes after administration of HNP‐2 in the transplantation chamber. Taken together, these results suggest that defensins could reverse a frequent immune alteration observed in cancer development.—Hubert, P., Herman, L., Maillard, C., Caberg, J‐H., Nikkels, A., Pierard, G., Foidart, J‐M., Noel, A., Boniver, J., Delvenne, P. Defensins induce the recruitment of dendritic cells in cervical human papillomavirus‐associated (pre)neoplastic lesions formed in vitro and transplanted in vivo. FASEB J. 21, 2765–2775 (2007)

Implementation of genomic arrays in prenatal diagnosis: The Belgian approach to meet the challenges

After their successful introduction in postnatal testing, genome-wide arrays are now rapidly replacing conventional karyotyping in prenatal diagnostics. While previous studies have demonstrated the advantages of this method, we are confronted with difficulties regarding the technology and the ethical dilemmas inherent to genomic arrays. These include indication for testing, array design, interpretation of variants and how to deal with variants of unknown significance and incidental findings. The experiences with these issues reported in the literature are most often from single centres. Here, we report on a national consensus approach how microarray is implemented in all genetic centres in Belgium. These recommendations are subjected to constant re-evaluation based on our growing experience and can serve as a useful tool for those involved in prenatal diagnosis.

Increased migration of Langerhans cells in response to HPV16 E6 and E7 oncogene silencing: role of CCL20

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intraepithelial lesions (SILs) show quantitative and functional alterations of Langerhans cells (LC). The infiltration of immature LC in the squamous epithelium is mainly controlled by Macrophage Inflammatory Protein 3α/CCL20. After having shown that CCL20 production is altered in HPV-transformed keratinocytes (KC), the possible role of HPV16 E6 and E7 viral oncoproteins in the reduced CCL20 levels observed in SILs was investigated by silencing HPV16 E6 and E7 oncogenes by RNA interference (siRNA). This treatment not only increased CCL20 secretion but also resulted in the modulation of NF-κB p50, p52 and p65 precursor localization. Moreover, silencing of E6 and E7 oncogenes in HPV16-transformed KC induced a significantly higher migratory capacity of LC in a Boyden chamber assay and in an in vitro formed (pre)neoplastic epithelium reminiscent of high-grade SILs. Anti-CCL20 neutralizing antibody experiments showed that the increased migration of LC is due to the re-expression of CCL20 in E6 and E7 siRNA transfected KC. These data suggest that HPV16 E6/E7-induced down-regulation of CCL20 observed during the cervical carcinogenesis may contribute to a diminished capacity of the immune system to control HPV infection.

Transforming Growth Factor-β1-Mediated Slug and Snail Transcription Factor Up-Regulation Reduces the Density of Langerhans Cells in Epithelial Metaplasia by Affecting E-Cadherin Expression

Epithelial metaplasia (EpM) is an acquired tissue abnormality resulting from the transformation of epithelium into another tissue with a different structure and function. This adaptative process is associated with an increased frequency of (pre)cancerous lesions. We propose that EpM is involved in cancer development by altering the expression of adhesion molecules important for cell-mediated antitumor immunity. Langerhans cells (LCs) are intraepithelial dendritic cells that initiate immune responses against viral or tumor antigens on both skin and mucosal surfaces. In the present study, we showed by immunohistology that the density of CD1a(+) LCs is reduced in EpM of the uterine cervix compared with native squamous epithelium and that the low number of LCs observed in EpM correlates with the down-regulation of cell-surface E-cadherin. We also demonstrated that transforming growth factor-beta1 is not only overexpressed in metaplastic tissues but also reduces E-cadherin expression in keratinocytes in vitro by inducing the promoter activity of Slug and Snail transcription factors. Finally, we showed that in vitro-generated LCs adhere poorly to keratinocytes transfected with either Slug or Snail DNA. These data suggest that transforming growth factor-beta1 indirectly reduces antigen-presenting cell density in EpM by affecting E-cadherin expression, which might explain the increased susceptibility of abnormal tissue differentiation to the development of cancer by the establishment of local immunodeficiency responsible for EpM tumorigenesis.

Somatic mosaicism underlies X-linked acrogigantism syndrome in sporadic male subjects

Somatic mosaicism has been implicated as a causative mechanism in a number of genetic and genomic disorders. X-linked acrogigantism (XLAG) syndrome is a recently characterized genomic form of pediatric gigantism due to aggressive pituitary tumors that is caused by submicroscopic chromosome Xq26.3 duplications that include GPR101 We studied XLAG syndrome patients (n= 18) to determine if somatic mosaicism contributed to the genomic pathophysiology. Eighteen subjects with XLAG syndrome caused by Xq26.3 duplications were identified using high-definition array comparative genomic hybridization (HD-aCGH). We noted that males with XLAG had a decreased log2ratio (LR) compared with expected values, suggesting potential mosaicism, whereas females showed no such decrease. Compared with familial male XLAG cases, sporadic males had more marked evidence for mosaicism, with levels of Xq26.3 duplication between 16.1 and 53.8%. These characteristics were replicated using a novel, personalized breakpoint junction-specific quantification droplet digital polymerase chain reaction (ddPCR) technique. Using a separate ddPCR technique, we studied the feasibility of identifying XLAG syndrome cases in a distinct patient population of 64 unrelated subjects with acromegaly/gigantism, and identified one female gigantism patient who had had increased copy number variation (CNV) threshold for GPR101 that was subsequently diagnosed as having XLAG syndrome on HD-aCGH. Employing a combination of HD-aCGH and novel ddPCR approaches, we have demonstrated, for the first time, that XLAG syndrome can be caused by variable degrees of somatic mosaicism for duplications at chromosome Xq26.3. Somatic mosaicism was shown to occur in sporadic males but not in females with XLAG syndrome, although the clinical characteristics of the disease were similarly severe in both sexes.