Co-Transplantation of Barcoded Lymphoid-Primed Multipotent (LMPP) and Common Lymphocyte (CLP) Progenitors Reveals a Major Contribution of LMPP to the Lymphoid Lineage.

T cells have the potential to maintain immunological memory and self-tolerance by recognizing antigens from pathogens or tumors. In pathological situations, failure to generate de novo T cells causes immunodeficiency resulting in acute infections and complications. Hematopoietic stem cells (HSC) transplantation constitutes a valuable option to restore proper immune function. However, delayed T cell reconstitution is observed compared to other lineages. To overcome this difficulty, we developed a new approach to identify populations with efficient lymphoid reconstitution properties. To this end, we use a DNA barcoding strategy based on the insertion into a cell chromosome of a lentivirus (LV) carrying a non-coding DNA fragment named barcode (BC). These will segregate through cell divisions and be present in cells' progeny. The remarkable characteristic of the method is that different cell types can be tracked simultaneously in the same mouse. Thus, we in vivo barcoded LMPP and CLP progenitors to test their ability to reconstitute the lymphoid lineage. Barcoded progenitors were co-grafted in immuno-compromised mice and their fate analyzed by evaluating the BC composition in transplanted mice. The results highlight the predominant role of LMPP progenitors for lymphoid generation and reveal valuable novel insights to be reconsidered in clinical transplantation assays.

The HLA-G immune checkpoint: a new immuno-stimulatory role for the α1-domain-deleted isoform.

The heterogeneity of cancer cells, in part maintained via the expression of multiple isoforms, introduces significant challenges in designing effective therapeutic approaches. In this regard, isoforms of the immune checkpoint HLA-G have been found in most of the tumors analyzed, such as ccRCC, the most common human renal malignancy. In particular, HLA-G∆α1, which is the only HLA-G isoform described that lacks the α1 extracellular domain, has been newly identified in ccRCC and now here in trophoblasts. Using a cellular model expressing HLA-G∆α1, we have uncovered its specific and overlapping functional roles, relative to the main HLA-G isoform, i.e., the full-length HLA-G1. We found that HLA-G∆α1 has several particular features: (i) although possessing the α3 domain, it does not associate with ß2-microglobulin; (ii) it may not present peptides to T cells due to absence of the peptide-binding groove; and (iii) it exerts immune-stimulatory activity towards peripheral blood NK and T cells, while all known isoforms of HLA-G are immune-inhibitory checkpoint molecules. Such immune-stimulatory properties of HLA-G∆α1 on the cytotoxic function of peripheral blood NK cells are individual dependent and are not exerted through the interaction with the known HLA-G receptor, ILT2. Importantly, we are faced here with a potential antitumor effect of an HLA-G isoform, opposed to the pro-tumor properties described for all other HLA-G isoforms, which should be taken into account in future therapeutic designs aimed at blocking this immune checkpoint.

Temporal Gene Expression Profiles Reflect the Dynamics of Lymphoid Differentiation.

Understanding the emergence of lymphoid committed cells from multipotent progenitors (MPP) is a great challenge in hematopoiesis. To gain deeper insight into the dynamic expression changes associated with these transitions, we report the quantitative transcriptome of two MPP subsets and the common lymphoid progenitor (CLP). While the transcriptome is rather stable between MPP2 and MPP3, expression changes increase with differentiation. Among those, we found that pioneer lymphoid genes such as Rag1, Mpeg1, and Dntt are expressed continuously from MPP2. Others, such as CD93, are CLP specific, suggesting their potential use as new markers to improve purification of lymphoid populations. Notably, a six-transcription factor network orchestrates the lymphoid differentiation program. Additionally, we pinpointed 24 long intergenic-non-coding RNA (lincRNA) differentially expressed through commitment and further identified seven novel forms. Collectively, our approach provides a comprehensive landscape of coding and non-coding transcriptomes expressed during lymphoid commitment.

HLA-G gene editing in tumor cell lines as a novel 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.W 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 HLA-G 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.

Role of the HLA-G immune checkpoint molecule in pregnancy.

The non-classical HLA class I molecule HLA-G is expressed in trophoblasts where it contributes to maternal-fetal tolerance. HLA-G has been implicated in the control of trophoblast invasion, uterine vascular remodeling, and maintenance of a local immunosuppressive state. Understanding HLA-G biology at the maternal-fetal interface is therefore a critical issue in reproduction. In this regard, we review here: (i) the effects of HLA-G on decidual leucocytes and stromal cells, (ii) the contribution of trogocytosis in HLA-G expression on decidual cells, (iii) its interaction with the ILT2, ILT4 and KIR2DL4 receptors, (iv) the link between HLA-G polymorphism and pregnancy disorders, and (v) the expression of newly-described HLA-G isoforms at the maternal-fetal interface.

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.

Comprehensive landscape of immune-checkpoints uncovered in clear cell renal cell carcinoma reveals new and emerging therapeutic targets.

Clear cell renal cell carcinoma (ccRCC) constitutes the most common renal cell carcinoma subtype and has long been recognized as an immunogenic cancer. As such, significant attention has been directed toward optimizing immune-checkpoints (IC)-based therapies. Despite proven benefits, a substantial number of patients remain unresponsive to treatment, suggesting that yet unreported, immunosuppressive mechanisms coexist within tumors and their microenvironment. Here, we comprehensively analyzed and ranked forty-four immune-checkpoints expressed in ccRCC on the basis of in-depth analysis of RNAseq data collected from the TCGA database and advanced statistical methods designed to obtain the group of checkpoints that best discriminates tumor from healthy tissues. Immunohistochemistry and flow cytometry confirmed and enlarged the bioinformatics results. In particular, by using the recursive feature elimination method, we show that HLA-G, B7H3, PDL-1 and ILT2 are the most relevant genes that characterize ccRCC. Notably, ILT2 expression was detected for the first time on tumor cells. The levels of other ligand-receptor pairs such as CD70:CD27; 4-1BB:4-1BBL; CD40:CD40L; CD86:CTLA4; MHC-II:Lag3; CD200:CD200R; CD244:CD48 were also found highly expressed in tumors compared to adjacent non-tumor tissues. Collectively, our approach provides a comprehensible classification of forty-four IC expressed in ccRCC, some of which were never reported before to be co-expressed in ccRCC. In addition, the algorithms used allowed identifying the most relevant group that best discriminates tumor from healthy tissues. The data can potentially assist on the choice of valuable immune-therapy targets which hold potential for the development of more effective anti-tumor treatments.

Intratumor heterogeneity of immune checkpoints in primary renal cell cancer: Focus on HLA-G/ILT2/ILT4.

The establishment and maintenance of anti-tumor immune responses are the objectives of cancer immunotherapy. Despite recent promising advances, the effectiveness of these approaches has been limited by the multiple immunosuppressive mechanisms developed by tumors (checkpoint). The aim of the present study was to demonstrate intratumor heterogeneity at the levels of immune escape strategies and tumor-host relationships. We focused on well-known checkpoints such as PD1/PDL1 and on a new checkpoint involving HLA-G and its receptors ILT2/ILT4. A prospective study was performed on 19 renal-cell carcinoma patients that were included during hospitalization for surgical tumor resection. Different areas of the tumor were collected for each patient and subjected to both immunohistochemical and flow cytometry analysis. Immune cells from peripheral blood were concomitantly analyzed for each patient. Our results show the heterogeneous expression of PD1/PDL1 and HLA-G/ILT in the various areas of the same tumor. Intratumor heterogeneity was found both at tumor cell and infiltrating immune cell levels. From a clinical point of view, this work highlights the functional redundancies of checkpoints and the need to adapt personalized poly-immunotherapy.

Novel landscape of HLA-G isoforms expressed 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. Human leukocyte antigen G (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 anticancer 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 intratumor 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: An Immune Checkpoint Molecule.

HLA-G is a molecule that was first known to confer protection to the fetus from destruction by the immune system of its mother, thus critically contributing to fetal-maternal tolerance. The first functional finding constituted the basis for HLA-G research and can be summarized as such: HLA-G, membrane-bound or soluble, strongly binds its inhibitory receptors on immune cells (NK, T, B, monocytes/dendritic cells), inhibits the functions of these effectors, and so induces immune inhibition. HLA-G function may therefore be beneficial because when expressed by a fetus or a transplant it protects them from rejection, or deleterious because when expressed by a tumor, it also protects it from antitumor immunity. This is the primary HLA-G function: that of a checkpoint molecule. Great work has been done in the past years to characterize HLA-G itself, its regulation, its functions and mechanisms of action, and its pathological relevance. We will review this here, focusing on transplantation and oncology because these pathological contexts have been studied the most and also because they best represent the two opposite sides of HLA-G: beneficial to be promoted, or deleterious to be blocked.

Single thrombopoietin dose alleviates hematopoietic stem cells intrinsic short- and long-term ionizing radiation damage. In vivo identification of anatomical cell expansion sites.

Hematopoietic stem cells (HSC) are essential for maintaining the integrity of complex and long-lived organisms. HSC, which are self-renewing, reconstitute the hematopoietic system through out life and facilitate long-term repopulation of myeloablated recipients. We have previously demonstrated that when mice are exposed to sublethal doses of ionizing radiation, subsets of the stem/progenitor compartment are affected. In this study we examine the role of thrombopoietin (TPO) on the regenerative capacities of HSC after irradiation and report the first demonstration of efficacy of a single injection of TPO shortly after in vivo exposure to ionizing radiation for reducing HSC injury and improving their functional outcome. Our results demonstrate that TPO treatment not only reduced the number of apoptotic cells but also induced a significant modification of their intrinsic characteristics. These findings were supported by transplantation assays with long-term HSC that were irradiated or unirradiated, TPO treated or untreated, in CD45.1/CD45.2 systems and by using luciferase-labeled HSC for direct bioluminescence imaging in living animals. Of particular importance, our data demonstrate the skull to be a highly favorable site for the TPO-induced emergence of hematopoietic cells after irradiation, suggesting a TPO-mediated relationship of primitive hematopoietic cells to an anatomical component. Together, the data presented here: provide novel findings about aspects of TPO action on stem cells, open new areas of investigation for therapeutic options in patients who are treated with radiation therapy, and show that early administration of a clinically suitable TPO-agonist counteracts the previously observed adverse effects.

The Notch Delta-4 ligand helps to maintain the quiescence and the short-term reconstitutive potential of haematopoietic progenitor cells through activation of a key gene network.

Understanding the role of Notch and its ligands within the different bone marrow niches could shed light on the mechanisms regulating haematopoietic progenitor cells (HPCs) maintenance and self-renewal. Here, we report that murine bone marrow HPCs activation by the vascular Notch Delta-4 ligand maintains a significant proportion of cells specifically in the G0 state. Furthermore, Delta-4/Notch pathway limits significantly the loss of the in vivo short-term reconstitutive potential upon transplantation of Delta-4 activated HPCs into lethally irradiated recipient mice. Both effects are directly correlated with the decrease of cell cycle genes transcription such as CYCLIN-D1, -D2, and -D3, and the upregulation of stemness related genes transcription such as BMI1, GATA2, HOXB4 and C-MYC. In addition, the transcriptional screening also highlights new downstream post-transcriptional factors, named PUMILIO1 and -2, as part of the stem signature associated with the Delta-4/Notch signalling pathway.

Arrayed lentiviral barcoding for quantification analysis of hematopoietic dynamics.

Our understanding of system dynamics of mixed cell populations in whole organisms has benefited from the advent of individual cell marking by nonarrayed DNA barcodes subsequently analyzed by high-throughput DNA sequencing. However, key limitations include statistical biases compromising quantification and the lack of applicability to deconvolute individual cell fate in vivo after pooling single cells differentially exposed to different conditions ex vivo. Here, we have derived an arrayed lentiviral library of DNA barcodes and obtained a proof-of-concept of its resolving capacity by quantifying hematopoietic regeneration after engraftment of mice with genetically modified autologous cells. This method has helped clarify and bridge the seemingly opposed clonal-succession and continuous-recruitment models of hematopoietic stem cell behavior and revealed that myeloid-lymphoid biases are common occurrences in steady-state hematopoiesis. Arrayed lentiviral barcoding should prove a versatile and powerful approach to deconvolute cell dynamics in vivo with applications in hematology, embryology, and cancer biology.

Transcriptome characterization uncovers the molecular response of hematopoietic cells to ionizing radiation.

Ionizing radiation causes rapid and acute suppression of hematopoietic cells that manifests as the hematopoietic syndrome. However, the roles of molecules and regulatory pathways induced in vivo by irradiation of different hematopoietic cells have not been completely elaborated. Using a strategy that combined different microarray bioinformatics tools, we identified gene networks that might be involved in the early response of hematopoietic cells radiation response in vivo. The grouping of similar time-ordered gene expression profiles using quality threshold clustering enabled the successful identification of common binding sites for 56 transcription factors that may be involved in the regulation of the early radiation response. We also identified novel genes that are responsive to the transformation-related protein 53; all of these genes were biologically validated in p53-transgenic null mice. Extension of the analysis to purified bone marrow cells including highly purified long-term hematopoietic stem cells, combined with functional classification, provided evidence of gene expression modifications that were largely unknown in this primitive population. Our methodology proved particularly useful for analyzing the transcriptional regulation of the complex ionizing radiation response of hematopoietic cells. Our data may help to elucidate the molecular mechanisms involved in tissue radiosensitivity and to identify potential targets for improving treatment in radiation emergencies.

Changes in transcriptome after in vivo exposure to ionising radiation reveal a highly specialised liver response.

PURPOSE: To identify transcriptional gene-networks involved in the early in vivo response of liver cells to radiation exposure and improve our understanding of the molecular processes responsible for tissue radiosensitivity. MATERIALS AND METHODS: Transcriptome variations of liver RNA samples were measured 3 hours post-irradiation using microarray technology. The results were confirmed and extended using real-time polymerase-chain-reaction (RT-PCR). RESULTS: We identified quantitative changes in the expression of 126 genes, most of which were observed for the first time. We show that some modifications, such as the upregulation of the cyclin-dependent kinase inhibitor 1A (Cdkn1A) gene, persisted for at least two months after the initial exposure. Other genes regulated by the transformation-related protein 53 (Trp53/p53) such as Bcl2-associated X protein (Bax) or etoposide-induced-2.4 (Ei24/PIG8) were not upregulated. Grouping differentially expressed genes into functional categories revealed that the primary response of liver cells to radiation exposure was the enhancement of oxidoreductase activity and inhibition of cell proliferation, involving cell cycle progression and apoptosis-related genes. CONCLUSIONS: The data provides evidence of gene expression modifications associated with the hepatic response to radiation exposure. One of the main differences observed with radiation-sensitive tissues such as the spleen was cell proliferation. The comparison of our data with transcriptome modifications in different biological models enabled the identification of networks of genes that might be co-regulated. Overall, our expression data revealed genes and cellular pathways that might help to improve our understanding of the molecular basis underlying tissue radiosensitivity and to identify possible targets for novel therapeutic strategies.

Phenotypic and functional changes induced in hematopoietic stem/progenitor cells after gamma-ray radiation exposure.

Ionizing radiation (IR) exposure causes rapid and acute bone marrow (BM) suppression that is reversible for nonlethal doses. Evidence is accumulating that IR can also provoke long-lasting residual hematopoietic injury. To better understand these effects, we analyzed phenotypic and functional changes in the stem/progenitor compartment of irradiated mice over a 10-week period. We found that hematopoietic stem cells (HSCs) identified by their repopulating ability continued to segregate within the Hoechst dye excluding "side population (SP)" early after IR exposure. However, transient phenotypic changes were observed within this cell population: Sca-1 (S) and c-Kit (K) expression levels were increased and severely reduced, respectively, with a concurrent increase in the proportion of SP(SK) cells positive for established indicators of the presence of HSCs: CD150 and CD105. Ten weeks after IR exposure, expression of Sca-1 and c-Kit at the SP cell surface returned to control levels, and BM cellularity of irradiated mice was restored. However, the c-Kit(+)Sca-1(+)Lin(-/low) (KSL) stem/progenitor compartment displayed major phenotypic modifications, including an increase and a severe decrease in the frequencies of CD150(+)Flk2(-) and CD150(-)Flk2(+) cells, respectively. CD150(+) KSL cells also showed impaired reconstituting ability, an increased tendency to apoptosis, and accrued DNA damage. Finally, 15 weeks after exposure, irradiated mice, but not age-matched controls, allowed engraftment and significant hematopoietic contribution from transplanted congenic HSCs without additional host conditioning. These results provide novel insight in our understanding of immediate and delayed IR-induced hematopoietic injury and highlight similarities between HSCs of young irradiated and old mice.

Modifying murine von Willebrand factor A1 domain for in vivo assessment of human platelet therapies.

The A1 domain of von Willebrand factor (VWF-A1) plays a crucial role in hemostasis and thrombosis by initiating platelet adhesion at sites of arterial injury through interactions with the platelet receptor glycoprotein Ib alpha (GPIbalpha). Here we report that murine VWF-A1 supports limited binding of human platelets. However, atomic models of GPIbalpha-VWF-A1 complexes identified an electrostatic 'hot-spot' that, when mutated in murine VWF-A1, switches its binding specificity from mouse to human GPIbalpha. Furthermore, mice expressing this mutant VWF-A1 display a bleeding phenotype that can be corrected by infusion of human platelets. Mechanistically, human platelets correct the phenotype by forming occlusive thrombi, an event that can be abrogated by blockade of GPIbalpha or by the preadministration of inhibitors of platelet activation or adhesion (clopidogrel (Plavix) and abciximab (ReoPro), respectively). Thus, by modifying a protein interface, we have generated a potential biological platform for preclinical screening of antithrombotics that specifically target human platelets.

Interrelation between polyploidization and megakaryocyte differentiation: a gene profiling approach.

Polyploidization is a part of the normal developmental process leading to platelet production during megakaryocyte (MK) differentiation. Ploidization is mainly involved in cell enlargement, but it is not clear whether gene expression is modified during MK ploidization. In this study, human MKs were grown from CD34(+) cells in the presence of thrombopoietin and sorted according to their ploidy level. A pangenomic microarray technique was applied to compare gene expression in 2N-, 4N-, 8N-, and 16N-sorted MKs. Using hierarchical clustering, we demonstrated that 2N and 4N MKs or 8N and 16N MKs are 2 different close populations with 105 discriminating genes. In the second approach, we determined the profile of genes that were continuously down- and up-regulated during polyploidization. Among the 100 down-regulated genes, 24 corresponded to genes involved in DNA replication and repair. The great majority of up-regulated genes corresponded to genes directly involved in platelet functions, such as genes encoding specific platelet glycoproteins and alpha-granule proteins, actin and microtubule cytoskeleton, factors involved in signaling, and transport proteins. Together, these results suggest that MK polyploidization per se does not regulate gene expression but is intrinsically included in the differentiation process.

Novel microarray-based method for estimating exposure to ionizing radiation.

Accurate estimation of the dose of ionizing radiation to which individuals have been exposed is critical for therapeutic treatment. We investigated whether gene expression profiles could be used to evaluate the dose received, thereby serving as a biological dosimeter. We used cDNA microarrays to monitor changes in gene expression profiles induced by ionizing radiation in mouse total blood. The subsets of genes best characterizing each dose were identified by resampling the original data set and calculating the intersection of the dose signatures. This analytical strategy minimizes the impact of potential genetic/epigenetic variation between mice and overcomes the bias in gene selection inherent to microarray technology. The significance of the identified signatures was evaluated by monitoring the type I error rate by in silico negative control simulation. Based on the distribution of the mean ratios of the selected probes, we were able to identify transcription profiles giving 83% to 100% correct estimation of the dose received by test mice, demonstrating that the selected probes could be used to determine the dose of radiation to which the animals had been exposed. This method could potentially be generalized to determine the level of exposure to other toxins and could be used to develop new related clinical applications.