The CXCL12/CXCR4 Signaling Pathway: A New Susceptibility Factor in Human Papillomavirus Pathogenesis.

The productive human papillomavirus (HPV) life cycle is tightly linked to the differentiation and cycling of keratinocytes. Deregulation of these processes and stimulation of cell proliferation by the action of viral oncoproteins and host cell factors underlies HPV-mediated carcinogenesis. Severe HPV infections characterize the wart, hypogammaglobulinemia, infection, and myelokathexis (WHIM) immunodeficiency syndrome, which is caused by gain-of-function mutations in the CXCR4 receptor for the CXCL12 chemokine, one of which is CXCR41013. We investigated whether CXCR41013 interferes in the HPV18 life cycle in epithelial organotypic cultures. Expression of CXCR41013 promoted stabilization of HPV oncoproteins, thus disturbing cell cycle progression and proliferation at the expense of the ordered expression of the viral genes required for virus production. Conversely, blocking CXCR41013 function restored virus production and limited HPV-induced carcinogenesis. Thus, CXCR4 and its potential activation by genetic alterations in the course of the carcinogenic process can be considered as an important host factor for HPV carcinogenesis.

OMIP-048 MC: Quantification of calcium sensors and channels expression in lymphocyte subsets by mass cytometry.

Calcium (Ca2+ ) signaling controls T-cell activation and functions. Ca2+ concentrations are locally detected and controlled by Ca2+ -sensors (STIM1 and 2 detecting the depletion from ER stores channels) and Ca2+ -channels (ORAI1-3 in the cell membrane and VDAC1 in the outer mitochondrial membrane). We first validated and titrated antibodies to assess the expression of these Ca2+ -sensors and -channels in human and murine cells, and further devised a 18-antibodies mass cytometry panel to characterize their expression in primary murine lymphocyte subsets.

Optimized protocol for 3D epithelial cultures supporting human papillomavirus replication.

Human papillomaviruses (HPVs) are commensal viruses with pathogenic potential. Their life cycle requires the proliferation and differentiation of keratinocytes (KCs) to form pluristratified epithelia. Based on the original organotypic epithelial raft cultures protocol, we provide an updated workflow to optimally generate pluristratified human epithelia supporting the complete HPV replicative life cycle, here called 3D full-thickness epithelial cultures (3Deps). We describe steps for HPV genome preparation, KC transfection, and dermal equivalent preparation. We then detail procedures for 3Deps culture, harvesting, and analysis.

Reprogramming of connexin landscape fosters fast gap junction intercellular communication in human papillomavirus-infected epithelia.

Human papillomaviruses (HPVs) are highly prevalent commensal viruses that require epithelial stratification to complete their replicative cycle. While HPV infections are most often asymptomatic, certain HPV types can cause lesions, that are usually benign. In rare cases, these infections may progress to non-replicative viral cycles associated with high HPV oncogene expression promoting cell transformation, and eventually cancer when not cleared by host responses. While the consequences of HPV-induced transformation on keratinocytes have been extensively explored, the impact of viral replication on epithelial homeostasis remains largely unexplored. Gap junction intercellular communication (GJIC) is critical for stratified epithelium integrity and function. This process is ensured by a family of proteins named connexins (Cxs), including 8 isoforms that are expressed in stratified squamous epithelia. GJIC was reported to be impaired in HPV-transformed cells, which was attributed to the decreased expression of the Cx43 isoform. However, it remains unknown whether and how HPV replication might impact on the expression of Cx isoforms and GJIC in stratified squamous epithelia. To address this question, we have used 3D-epithelial cell cultures (3D-EpCs), the only model supporting the productive HPV life cycle. We report a transcriptional downregulation of most epithelial Cx isoforms except Cx45 in HPV-replicating epithelia. At the protein level, HPV replication results in a reduction of Cx43 expression while that of Cx45 increases and displays a topological shift toward the cell membrane. To quantify GJIC, we pioneered quantitative gap-fluorescence loss in photobleaching (FLIP) assay in 3D-EpCs, which allowed us to show that the reprogramming of Cx landscape in response to HPV replication translates into accelerated GJIC in living epithelia. Supporting the pathophysiological relevance of our observations, the HPV-associated Cx43 and Cx45 expression pattern was confirmed in human cervical biopsies harboring HPV. In conclusion, the reprogramming of Cx expression and distribution in HPV-replicating epithelia fosters accelerated GJIC, which may participate in epithelial homeostasis and host immunosurveillance.

MLL-ENL leukemia burden initiated in femoral diaphysis and preceded by mature B-cell depletion.

BACKGROUND: Molecular and cellular events that resulted in leukemia development are well characterized but initial engraftment and proliferation of leukemic cells in bone marrow and early modifications of the bone marrow microenvironment induced by engrafted leukemic cells remain to be clarified. DESIGN AND METHODS: After retro-orbital injection of 1,000 leukemic cells expressing Mixed Lineage Leukemia-Eleven Nineteen Leukemia fusion protein in non-conditioned syngenic mice, kinetics of leukemic burden and alterations of femoral hematopoietic populations were followed using an in vivo confocal imaging system and flow cytometry. RESULTS: Three days after injection, 5% of leukemic cells were found in femurs. Little proliferation of engrafted leukemic cells could then be detected for more than two weeks while the number of femoral leukemic cells remained stable. Twenty days after injection, leukemic cells preferentially proliferated in femoral diaphysis where they formed clusters on the surface of blood vessels and bone. B220(+) lymphoid cells were found near these leukemic cell clusters and this association is correlated with a decreased number of femoral B220(+)IgM(+) cells. Increasing the number of injected leukemic cells or conditioning recipient mice with γ-irradiation resulted in leukemic cell development in diaphysis and knee. Competition experiments indicate that proliferation but not engraftment is a rate-limiting factor of leukemic cells spreading in diaphysis. Finally, 30 days after injection leukemia developed. CONCLUSIONS: After retro-orbital injection of 1,000 leukemic cells expressing Mixed Lineage Leukemia-Eleven Nineteen Leukemia into syngenic mice, leukemic cell burden preferentially initiates in femoral diaphysis and is preceded by changes of femoral B-lymphoid populations.

Multi-Tissue Characterization of GILZ Expression in Dendritic Cell Subsets at Steady State and in Inflammatory Contexts.

Dendritic cells (DCs) are key players in the control of tolerance and immunity. Glucocorticoids (GCs) are known to regulate DC function by promoting their tolerogenic differentiation through the induction of inhibitory ligands, cytokines, and enzymes. The GC-induced effects in DCs were shown to critically depend on increased expression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ). GILZ expression levels were further shown to control antigen-presenting cell function, as well as T-cell priming capacity of DCs. However, the pattern of GILZ expression in DC subsets across tissues remains poorly described, as well as the modulation of its expression levels in different pathological settings. To fill in this knowledge gap, we conducted an exhaustive analysis of GILZ relative expression levels in DC subsets from various tissues using multiparametric flow cytometry. This study was performed at steady state, in the context of acute as well as chronic skin inflammation, and in a model of cancer. Our results show the heterogeneity of GILZ expression among DC subsets as well as the complexity of its modulation, that varies in a cell subset- and context-specific manner. Considering the contribution of GILZ in the control of DC functions and its potential as an immune checkpoint in cancer settings, these results are of high relevance for optimal GILZ targeting in therapeutic strategies.

Differential activity and selectivity of N-terminal modified CXCL12 chemokines at the CXCR4 and ACKR3 receptors.

Chemokines play critical roles in numerous physiologic and pathologic processes through their action on seven-transmembrane (TM) receptors. The N-terminal domain of chemokines, which is a key determinant of signaling via its binding within a pocket formed by receptors' TM helices, can be the target of proteolytic processing. An illustrative case of this regulatory mechanism is the natural processing of CXCL12 that generates chemokine variants lacking the first two N-terminal residues. Whereas such truncated variants behave as antagonists of CXCR4, the canonical G protein-coupled receptor of CXCL12, they are agonists of the atypical chemokine receptor 3 (ACKR3/CXCR7), suggesting the implication of different structural determinants in the complexes formed between CXCL12 and its two receptors. Recent analyses have suggested that the CXCL12 N-terminus first engages the TM helices of ACKR3 followed by the receptor N-terminus wrapping around the chemokine core. Here we investigated the first stage of ACKR3-CXCL12 interactions by comparing the activity of substituted or N-terminally truncated variants of CXCL12 toward CXCR4 and ACKR3. We showed that modification of the first two N-terminal residues of the chemokine (K1R or P2G) does not alter the ability of CXCL12 to activate ACKR3. Our results also identified the K1R variant as a G protein-biased agonist of CXCR4. Comparative molecular dynamics simulations of the complexes formed by ACKR3 either with CXCL12 or with the P2G variant identified interactions between the N-terminal 2-4 residues of CXCL12 and a pocket formed by receptor's TM helices 2, 6, and 7 as critical determinants for ACKR3 activation.

Paternal Insulin-like Growth Factor 2 (Igf2) Regulates Stem Cell Activity During Adulthood.

Insulin-like Growth Factor 2 (IGF2) belongs to the IGF/Insulin pathway, a highly conserved evolutionarily network that regulates growth, aging and lifespan. Igf2 is highly expressed in the embryo and in cancer cells. During mouse development, Igf2 is expressed in all sites where hematopoietic stem cells (HSC) successively expand, then its expression drops at weaning and becomes undetectable when adult HSC have reached their niches in bones and start to self-renew. In the present study, we aim to discover the role of IGF2 during adulthood. We show that Igf2 is specifically expressed in adult HSC and we analyze HSC from adult mice deficient in Igf2 transcripts. We demonstrate that Igf2 deficiency avoids the age-related attrition of the HSC pool and that Igf2 is necessary for tissue homeostasis and regeneration. Our study reveals that the expression level of Igf2 is critical to maintain the balance between stem cell self-renewal and differentiation, presumably by regulating the interaction between HSC and their niche. Our data have major clinical interest for transplantation: understanding the changes in adult stem cells and their environments will improve the efficacy of regenerative medicine and impact health- and life-span.

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

Adult hematopoiesis is regulated by TIF1γ, a repressor of TAL1 and PU.1 transcriptional activity.

Crosstalk between transcription factors and cytokines precisely regulates tissue homeostasis. Transcriptional intermediary factor 1γ (TIF1γ) regulates vertebrate hematopoietic development, can control transcription elongation, and is a component of the TGF-ß signaling pathway. Here we show that deletion of TIF1γ in adult hematopoiesis is compatible with life and long-term maintenance of essential blood cell lineages. However, loss of TIF1γ results in deficient long-term hematopoietic stem cell (LT-HSC) transplantation activity, deficient short-term HSC (ST-HSC) bone marrow retention, and priming ST-HSCs to myelomonocytic lineage. These defects are hematopoietic cell-autonomous, and priming of TIF1γ-deficient ST-HSCs can be partially rescued by wild-type hematopoietic cells. TIF1γ can form complexes with TAL1 or PU.1-two essential DNA-binding proteins in hematopoiesis-occupy specific subsets of their DNA binding sites in vivo, and repress their transcriptional activity. These results suggest a regulation of adult hematopoiesis through TIF1γ-mediated transcriptional repression of TAL1 and PU.1 target genes.