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

Characterization of thrombopoietin (TPO)-responsive progenitor cells in adult mouse bone marrow with in vivo megakaryocyte and erythroid potential

Hematopoietic progenitor cells are the progeny of hematopoietic stem cells that coordinate the production of precise numbers of mature blood cells of diverse functional lineages. Identification of cell-surface antigen expression associated with hematopoietic lineage restriction has allowed prospective isolation of progenitor cells with defined hematopoietic potential. To clarify further the cellular origins of megakaryocyte commitment, we assessed the in vitro and in vivo megakaryocyte and platelet potential of defined progenitor populations in the adult mouse bone marrow. We show that megakaryocytes arise from CD150+ bipotential progenitors that display both platelet- and erythrocyte-producing potential in vivo and that can develop from the Flt3− fraction of the pregranulocyte-macrophage population. We define a bipotential erythroid-megakaryocyte progenitor population, the CD150+CD9loendoglinlo fraction of Lin−cKit+IL7 receptor alpha−FcγRII/IIIloSca1− cells, which contains the bulk of the megakaryocyte colony-forming capacity of the bone marrow, including bipotential megakaryocyte-erythroid colony-forming capacity, and can generate both erythrocytes and platelets efficiently in vivo. This fraction is distinct from the CD150+CD9hiendoglinlo fraction, which contains bipotential precursors with characteristics of increased megakaryocytic maturation, and the CD150+CD9loendoglinhi fraction, which contains erythroid lineage-committed cells. Finally, we demonstrate that bipotential erythroid-megakaryocyte progenitor and CD150+CD9hiendoglinlo cells are TPO-responsive and that the latter population specifically expands in the recovery from thrombocytopenia induced by anti-platelet serum

Dataset for: Novel landscape of HLA-G isoforms in clear cell renal cell carcinoma patients

Immune-checkpoints are powerful inhibitory molecules that promote tumor survival. Their blockade is now recognized as providing effective therapeutic benefit against cancer. HLA-G, a recently identified immune checkpoint, has been detected in many types of primary tumors and metastases, in malignant effusions as well as on tumor-infiltrating cells, particularly in patients with clear cell renal cell carcinoma (ccRCC). Here, in order to define a possible anti-cancer therapy, we used a molecular approach based on an unbiased strategy that combines transcriptome determination and immunohistochemical labeling, to analyze in-depth, the HLA-G isoforms expressed in these tumors. We found that the expression of HLA-G is highly variable among tumors and distinct areas of the same tumor, testifying a marked inter- and intra-tumor heterogeneity. Moreover, our results generate an inventory of novel HLA-G isoforms which includes spliced forms that have an extended 5’-region and lack the transmembrane and alpha-1 domains. So far, these isoforms could not be detected by any method available and their assessment may improve the procedure by which tumors are analyzed. Collectively, our approach provides the first extensive portrait of HLA-G in ccRCC and reveals data that should prove suitable for the tailoring of future clinical applications

HLA-G gene editing: a novel therapeutic alternative in cancer immunotherapy

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial. In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLAG was reached to different degrees, including complete silencing. Most importantly, HLA-G – cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.Facultad de Ciencias MédicasComisión de Investigaciones Científicas de la provincia de Buenos Aire