HLA-G/LILRBs: A Cancer Immunotherapy Challenge.

Despite some success, many patients do not benefit from immunotherapy. New strategies to improve clinical efficacy include identification of novel immune-checkpoint (IC) targets or a combination of immunotherapy with antiangiogenic treatments. Here, we propose the therapeutic use of IC, HLA-G/LILRB, and explore its enhanced synergistic antitumor activity when combined with antiangiogenic therapies

The immune-checkpoint HLA-G/ILT4 is involved in the regulation of VEGF expression in clear cell renal cell carcinoma

Background: Clear cell renal cell carcinoma (ccRCC), the most aggressive renal cancer, is characterized by early lymph node metastases and bad prognosis. Most therapies targeting advanced or metastatic ccRCC are based, as first-line treatment, on the administration of the vascular endothelial growth factor (VEGF) neutralizing antibody termed Bevacizumab. Despite proven benefits, the expected results were not obtained for the majority of patients. The possibility that an intricate interplay between angiogenesis and immune-checkpoints might exist lead us to evaluate tumor angiogenesis, by means of VEGF expression together with the immune checkpoint HLA-G/ILT4. Methods: Tumor specimens were obtained from patients from two separate cohorts: One from “Evita Pueblo” Hospital from Berazategui, (Buenos Aires, Argentina) and the second includes patients surgically operated at the Urology Department of Saint-Louis Hospital (Paris, France) with a confirmed ccRCC diagnosis. Immunohistochemistry was performed with specific antibodies directed against HLA-G, VEGF-A, VEGF-C, D240, CD34, ILT4 and Ca-IX. In addition, gene expression levels were measured in a cell line derived from a ccRCC patient by semi-quantitative RT-PCR. Results: Our results show that the highly vascularized tumors of ccRCC patients express high levels of VEGF and the immune-checkpoint HLA-G. In addition, ILT4, one of the HLA-G receptors, was detected on macrophages surrounding tumor cells, suggesting the generation of an immune-tolerant microenvironment that might favor tumorigenesis. Notably, RT-qPCR analysis provided the first evidence on the transcriptional relationship between HLA-G/ILT4 and the VEGF family. Namely, in the presence of HLA-G or ILT4, the levels of VEGF-A are diminished whereas those of VEGF-C are increased. Conclusions: In an effort to find new therapeutic molecules and fight against metastasis dissemination associated with the poor survival rates of ccRCC patients, these findings provide the rationale for co-targeting angiogenesis and the immune checkpoint HLA-G.Facultad de Ciencias Médica

Genetic and Phenotypic Modifications of Hematopoietic Stem Cells in Response to Ionizing Radiation Exposure

Hematopoietic stem cells (HSC) constitute a rare population within the bone marrow (BM) which actively maintains continuous production of all mature blood cell-lineages throughout life. They are finely regulated to respond rapidly and specifically to modifications of the number of circulating cells or to environmental modifications such as radiation exposure. Although a marked sensibility to radiation exposure of these cells has been reported, little is known about the underlying mechanisms. To understand at the molecular level the cell-dependent IR response, phenotypical and transcriptional changes occurring in stem cells were monitored following exposure to total body irradiation at 2Gy, 3Gy, 6Gy, in a kinetic study from 1 hour up to 60 days. Different stem cell populations were isolated using combinations of surface molecules such as Lin-/low Sca+ C-Kit+ (LSK) with or without CD150+ and CD135+. Our results show that cells with this phenotype were completely eradicated 48h after radiation exposure. Microarray analysis showed that as early as 1 hour after radiation exposure stem cells elicit a specific damage response mostly triggered by the modulation of apoptotic genes. Many genes identified were never described before as playing a role in the IR response. Moreover, QT clustering of expression profiles and subsequent promoter analysis of co-clustered genes reveal several novel P53 co-regulated genes. The induction of these genes after radiation exposure was impaired in mutant mice deficient for P53. Two months after 3 Gy or 6 Gy TBI, the BM cellularity was back to normal but the percentage of LSK cells was reduced and the proportion of LSK subsets expressing CD150 and/or CD135 was modified in a dose-dependant manner. Moreover, HSC presented a major defect in their long-term reconstitution potential that aroused from a blockage in their differentiation potential. This blockage can be partially abrogated by the administration of thrombopoietin. Novel actors in this signalling pathway were identified and will be presented. These results demonstrate that irradiation, on the one hand, kill a vast majority of cycling cells in the BM by the early onset of genetic network of apoptotic genes, and on the other hand affect quiescent HSC in a cell-autonomous but reversible manner. Altogether our approach help producing a more complete knowledge of the molecular mechanisms of the stem cell radiation response by unraveling molecular networks and finding novel and specific molecules involved in this process. This information might facilitate the derivation of clinically useful targets for patient benefit

Transcriptional diversity and developmental potential of early hematopoietic progenitors revealed by cellular barcoding and transcriptome-wide profiling

International audienceBackground: One of the main complications of anti-cancer therapies or bone marrow transplantation protocols is their deleterious effect on the blood system, leading to prolonged neutropenia and increased risk for infections. Manipulating hematopoietic stem cells differentiation pathways to favor production of specific lineage-committed progenitors might optimize blood recovery. Aims: In this study we aimed 1) to determine and quantify the contribution of medullary progenitor populations (MPP) to the repopulation of the T cell pathway using the barcode cellular labelling strategy that we have previously developed and 2) to decipher the heterogeneity of these MPP at the transcriptional level. Methods: Three different MPP subsets, of the following phenotype: VCAM1+Flt3 (MPP1); VCAM1-Flt3+ (MPP2) and VCAM-1Flt3+ILR7 (CLP), were tagged with different barcodes carried by a collection of lentivirus and transplanted in mice. Barcoded cells in recipient mice were analyzed by flow cytometry. For whole transcriptome strand-specific sequencing, three biological replicates, per cell population, were sequenced at high depth of coverage (2 x 120 million reads). Results: The results allowed the in vivo dynamic tracking of the progeny of the barcoded progenitors in transplanted recipients. Moreover, transcriptome-wide profiling allowed to identify, by cluster analysis of RNAseq profiles together with gene ontology annotation, unique co-expressed markers for the prospective isolation of these populations. Unsupervised classification correctly classified reference surface markers, currently used to purify progenitors, which validate our bioinformatic methodology. Transcriptional regulation of these cell surface markers was further assess by searching for co-expressed transcription factors and enriched binding sites in their promoters. Their grouping enabled to establish undescribed regulatory networks, specific to each progenitor cell. Summary/Conclusions: Collectively, the cellular barcoding tool and the molecular changes observed at RNA and functional levels as they occur in vivo in the context of physiologic commitment processes, highlighted data that contribute to a deeper understanding of the dynamic of T-lineage differentiation and the lineage restriction process