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.

Human Keratinocytes Inhibit CD4+ T-Cell Proliferation through TGFB1 Secretion and Surface Expression of HLA-G1 and PD-L1 Immune Checkpoints.

Human skin protects the body against infection and injury. This protection involves immune and epithelial cells, but their interactions remain largely unknown. Here, we show that cultured epidermal keratinocytes inhibit allogenic CD4+ T-cell proliferation under both normal and inflammatory conditions. Inhibition occurs through the secretion of soluble factors, including TGFB1 and the cell-surface expression of HLA-G1 and PD-L1 immune checkpoints. For the first time, we here describe the expression of the HLA-G1 protein in healthy human skin and its role in keratinocyte-driven tissue immunomodulation. The overexpression of HLA-G1 with an inducible vector increased the immunosuppressive properties of keratinocytes, opening up perspectives for their use in allogeneic settings for cell therapy.

Cord blood-endothelial colony forming cells are immunotolerated and participate at post-ischemic angiogenesis in an original dorsal chamber immunocompetent mouse model.

BACKGROUND: Cardiovascular diseases are the main cause of morbidity and mortality worldwide. Restoring blood supply to ischemic tissues is an essential goal for the successful treatment of these diseases. Growth factor or gene therapy efficacy remains controversial, but stem cell transplantation is emerging as an interesting approach to stimulate angiogenesis. Among the different stem cell populations, cord blood-endothelial progenitor cells (CB-EPCs) and more particularly cord blood-endothelial progenitor cell-derived endothelial colony forming cells (CB-ECFCs) have a great proliferative potential without exhibiting signs of senescence. Even if it was already described that CB-ECFCs were able to restore blood perfusion in hind-limb ischemia in an immunodeficient mouse model, until now, the immunogenic potential of allogenic CB-ECFCs remains controversial. Therefore, our objectives were to evaluate the immune tolerance potency of CB-ECFCs and their capacity to restore a functional vascular network under ischemic condition in immunocompetent mice. METHODS: In vitro, the expression and secretion of immunoregulatory markers (HLA-G, IL-10, and TGF-ß1) were evaluated on CB-ECFCs. Moreover, CB-ECFCs were co-cultured with activated peripheral blood mononuclear cells (PBMCs) for 6 days. PBMC proliferation was evaluated by [3H]-thymidine incorporation on the last 18 h. In vivo, CB-ECFCs were administered in the spleen and muscle of immunocompetent mice. Tissues were collected at day 14 after surgery. Finally, CB-ECFCs were injected intradermally in C57BL/6JRj mice close to ischemic macrovessel induced by thermal cauterization. Mice recovered until day 5 and were imaged, twice a week until day 30. RESULTS: Firstly, we demonstrated that CB-ECFCs expressed HLA-G, IL-10, and TGF-ß1 and secreted IL-10 and TGF-ß1 and that they could display immunosuppressive properties in vitro. Secondly, we showed that CB-ECFCs could be tolerated until 14 days in immunocompetent mice. Thirdly, we revealed in an original ischemic model of dorsal chamber that CB-ECFCs were integrated in a new functional vascular network. CONCLUSION: These results open up new perspectives about using CB-ECFCs as an allogeneic cell therapy product and gives new impulse to the treatment of cardiovascular diseases.

Are the Immune Properties of Mesenchymal Stem Cells from Wharton's Jelly Maintained during Chondrogenic Differentiation?

BACKGROUND: Umbilical mesenchymal stem/stromal cells (MSCs), and especially those derived from Wharton's jelly (WJ), are a promising engineering tool for tissue repair in an allogeneic context. This is due to their differentiation capacity and immunological properties, like their immunomodulatory potential and paracrine activity. Hence, these cells may be considered an Advanced Therapy Medicinal Product (ATMP). The purpose of this work was to differentiate MSCs from WJ (WJ-MSCs) into chondrocytes using a scaffold and to evaluate, in vitro, the immunomodulatory capacities of WJ-MSCs in an allogeneic and inflammatory context, mimicked by IFN-γ and TNF-α priming during the chondrogenic differentiation. METHODS: Scaffolds were made from hydrogel composed by alginate enriched in hyaluronic acid (Alg/HA). Chondrogenic differentiation, immunological function, phenotype expression, but also secreted soluble factors were the different parameters followed during 28 days of culture. RESULTS: During chondrocyte differentiation, even in an allogeneic context, WJ-MSCs remained unable to establish the immunological synapse or to induce T cell alloproliferation. Moreover, interestingly, paracrine activity and functional immunomodulation were maintained during cell differentiation. CONCLUSION: These results show that WJ-MSCs remained hypoimmunogenic and retained immunomodulatory properties even when they had undergone chondrocyte differentiation.

Habitat and taxon as driving forces of carbohydrate catabolism in marine heterotrophic bacteria: example of the model algae-associated bacterium Zobellia galactanivorans DsijT.

The marine flavobacterium Zobellia galactanivorans DsijT was isolated from a red alga and by now constitutes a model for studying algal polysaccharide bioconversions. We present an in-depth analysis of its complete genome and link it to physiological traits. Z. galactanivorans exhibited the highest gene numbers for glycoside hydrolases, polysaccharide lyases and carbohydrate esterases and the second highest sulfatase gene number in a comparison to 125 other marine heterotrophic bacteria (MHB) genomes. Its genome contains 50 polysaccharide utilization loci, 22 of which contain sulfatase genes. Catabolic profiling confirmed a pronounced capacity for using algal polysaccharides and degradation of most polysaccharides could be linked to dedicated genes. Physiological and biochemical tests revealed that Z. galactanivorans stores and recycles glycogen, despite loss of several classic glycogen-related genes. Similar gene losses were observed in most Flavobacteriia, suggesting presence of an atypical glycogen metabolism in this class. Z. galactanivorans features numerous adaptive traits for algae-associated life, such as consumption of seaweed exudates, iodine metabolism and methylotrophy, indicating that this bacterium is well equipped to form profitable, stable interactions with macroalgae. Finally, using statistical and clustering analyses of the MHB genomes we show that their carbohydrate catabolism correlates with both taxonomy and habitat.

Mesenchymal Stem Cells Derived from Human Bone Marrow and Adipose Tissue Maintain Their Immunosuppressive Properties After Chondrogenic Differentiation: Role of HLA-G.

Mesenchymal stem cells (MSC) have emerged as alternative sources of stem cells for regenerative medicine because of their multipotency and strong immune-regulatory properties. Also, human leukocyte antigen G (HLA-G) is an important mediator of MSC-mediated immunomodulation. However, it is unclear whether MSC retain their immune-privileged potential after differentiation. As promising candidates for cartilage tissue engineering, the immunogenic and immunomodulatory properties of chondro-differentiated MSC (chondro-MSC) require in-depth exploration. In the present study, we used the alginate/hyaluronic acid (Alg/HA) hydrogel scaffold and induced both bone marrow- and adipose tissue-derived MSC into chondrocytes in three-dimensional condition. Then, MSC before and after chondrocyte differentiation were treated or not with interferon γ and tumor necrosis factor α mimicking inflammatory conditions and were compared side by side using flow cytometry, mixed lymphocyte reaction, and immunostaining assays. Results showed that chondro-MSC were hypoimmunogenic and could exert immunosuppression on HLA-mismatched peripheral blood mononuclear cells as well as undifferentiated MSC did. This alloproliferation inhibition mediated by MSC or chondro-MSC was dose dependent. Meanwhile, chondro-MSC exerted inhibition on natural killer cell-mediated cytolysis. Also, we showed that HLA-G expression was upregulated in chondro-MSC under hypoxia context and could be boosted in allogenic settings. Besides, the Alg/HA hydrogel scaffold was hypoimmunogenic and its addition for supporting MSC chondrocyte differentiation did not modify the immune properties of MSC. Finally, considering their chondro-regenerative potential and their retained immunosuppressive capacity, MSC constitute promising allogenic sources of stem cells for cartilage repair.

Life in an arsenic-containing gold mine: genome and physiology of the autotrophic arsenite-oxidizing bacterium rhizobium sp. NT-26.

Arsenic is widespread in the environment and its presence is a result of natural or anthropogenic activities. Microbes have developed different mechanisms to deal with toxic compounds such as arsenic and this is to resist or metabolize the compound. Here, we present the first reference set of genomic, transcriptomic and proteomic data of an Alphaproteobacterium isolated from an arsenic-containing goldmine: Rhizobium sp. NT-26. Although phylogenetically related to the plant-associated bacteria, this organism has lost the major colonizing capabilities needed for symbiosis with legumes. In contrast, the genome of Rhizobium sp. NT-26 comprises a megaplasmid containing the various genes, which enable it to metabolize arsenite. Remarkably, although the genes required for arsenite oxidation and flagellar motility/biofilm formation are carried by the megaplasmid and the chromosome, respectively, a coordinate regulation of these two mechanisms was observed. Taken together, these processes illustrate the impact environmental pressure can have on the evolution of bacterial genomes, improving the fitness of bacterial strains by the acquisition of novel functions.

HLA-G1 and HLA-G5 active dimers are present in malignant cells and effusions: the influence of the tumor microenvironment.

Dimers of the nonclassical HLA-G class I molecule have recently been shown to be active structures that mediate inhibition of NK-cell cytotoxic activity through interaction with the immunoglobulin-like transcript (ILT)-2 inhibitory receptor. However, this has only been proven in trophoblasts and HLA-G transfectants. Here, we document for the first time the existence of HLA-G dimers in cancer. Indeed, we identified both surface and soluble HLA-G dimers in tumor cells and malignant ascites respectively. Interestingly, factors from the tumor microenvironment, such as interferons, enhanced the formation of HLA-G dimers and increased the protection of tumors from NK cell-mediated lysis. These data emphasize the impact of HLA-G conformation on its efficiency at inhibiting the antitumor response and thus favoring tumor progression. In view of these results, the effect of the tumor microenvironment on upregulation of HLA-G function deserves particular attention when designing cancer immunotherapy protocols.

Fine-scale evolution: genomic, phenotypic and ecological differentiation in two coexisting Salinibacter ruber strains.

Genomic and metagenomic data indicate a high degree of genomic variation within microbial populations, although the ecological and evolutive meaning of this microdiversity remains unknown. Microevolution analyses, including genomic and experimental approaches, are so far very scarce for non-pathogenic bacteria. In this study, we compare the genomes, metabolomes and selected ecological traits of the strains M8 and M31 of the hyperhalophilic bacterium Salinibacter ruber that contain ribosomal RNA (rRNA) gene and intergenic regions that are identical in sequence and were simultaneously isolated from a Mediterranean solar saltern. Comparative analyses indicate that S. ruber genomes present a mosaic structure with conserved and hypervariable regions (HVRs). The HVRs or genomic islands, are enriched in transposases, genes related to surface properties, strain-specific genes and highly divergent orthologous. However, the many indels outside the HVRs indicate that genome plasticity extends beyond them. Overall, 10% of the genes encoded in the M8 genome are absent from M31 and could stem from recent acquisitions. S. ruber genomes also harbor 34 genes located outside HVRs that are transcribed during standard growth and probably derive from lateral gene transfers with Archaea preceding the M8/M31 divergence. Metabolomic analyses, phage susceptibility and competition experiments indicate that these genomic differences cannot be considered neutral from an ecological perspective. The results point to the avoidance of competition by micro-niche adaptation and response to viral predation as putative major forces that drive microevolution within these Salinibacter strains. In addition, this work highlights the extent of bacterial functional diversity and environmental adaptation, beyond the resolution of the 16S rRNA and internal transcribed spacers regions.

Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity.

BACKGROUND: While the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study. RESULTS: The two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms. CONCLUSION: Overall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events.

Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus.

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a "mycobacterial" gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the "non mycobacterial" factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients.

Genome sequence of the beta-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia.

We report the first complete genome sequence of a beta-proteobacterial nitrogen-fixing symbiont of legumes, Cupriavidus taiwanensis LMG19424. The genome consists of two chromosomes of size 3.42 Mb and 2.50 Mb, and a large symbiotic plasmid of 0.56 Mb. The C. taiwanensis genome displays an unexpected high similarity with the genome of the saprophytic bacterium C. eutrophus H16, despite being 0.94 Mb smaller. Both organisms harbor two chromosomes with large regions of synteny interspersed by specific regions. In contrast, the two species host highly divergent plasmids, with the consequence that C. taiwanensis is symbiotically proficient and less metabolically versatile. Altogether, specific regions in C. taiwanensis compared with C. eutrophus cover 1.02 Mb and are enriched in genes associated with symbiosis or virulence in other bacteria. C. taiwanensis reveals characteristics of a minimal rhizobium, including the most compact (35-kb) symbiotic island (nod and nif) identified so far in any rhizobium. The atypical phylogenetic position of C. taiwanensis allowed insightful comparative genomics of all available rhizobium genomes. We did not find any gene that was both common and specific to all rhizobia, thus suggesting that a unique shared genetic strategy does not support symbiosis of rhizobia with legumes. Instead, phylodistribution analysis of more than 200 Sinorhizobium meliloti known symbiotic genes indicated large and complex variations of their occurrence in rhizobia and non-rhizobia. This led us to devise an in silico method to extract genes preferentially associated with rhizobia. We discuss how the novel genes we have identified may contribute to symbiotic adaptation.

Being pathogenic, plastic, and sexual while living with a nearly minimal bacterial genome.

Mycoplasmas are commonly described as the simplest self-replicating organisms, whose evolution was mainly characterized by genome downsizing with a proposed evolutionary scenario similar to that of obligate intracellular bacteria such as insect endosymbionts. Thus far, analysis of mycoplasma genomes indicates a low level of horizontal gene transfer (HGT) implying that DNA acquisition is strongly limited in these minimal bacteria. In this study, the genome of the ruminant pathogen Mycoplasma agalactiae was sequenced. Comparative genomic data and phylogenetic tree reconstruction revealed that approximately 18% of its small genome (877,438 bp) has undergone HGT with the phylogenetically distinct mycoides cluster, which is composed of significant ruminant pathogens. HGT involves genes often found as clusters, several of which encode lipoproteins that usually play an important role in mycoplasma-host interaction. A decayed form of a conjugative element also described in a member of the mycoides cluster was found in the M. agalactiae genome, suggesting that HGT may have occurred by mobilizing a related genetic element. The possibility of HGT events among other mycoplasmas was evaluated with the available sequenced genomes. Our data indicate marginal levels of HGT among Mycoplasma species except for those described above and, to a lesser extent, for those observed in between the two bird pathogens, M. gallisepticum and M. synoviae. This first description of large-scale HGT among mycoplasmas sharing the same ecological niche challenges the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution. The latter clearly differs from that of other bacteria with small genomes, particularly obligate intracellular bacteria that are isolated within host cells. Consequently, mycoplasmas are not only able to subvert complex hosts but presumably have retained sexual competence, a trait that may prevent them from genome stasis and contribute to adaptation to new hosts.

Genome characteristics of facultatively symbiotic Frankia sp. strains reflect host range and host plant biogeography.

Soil bacteria that also form mutualistic symbioses in plants encounter two major levels of selection. One occurs during adaptation to and survival in soil, and the other occurs in concert with host plant speciation and adaptation. Actinobacteria from the genus Frankia are facultative symbionts that form N(2)-fixing root nodules on diverse and globally distributed angiosperms in the "actinorhizal" symbioses. Three closely related clades of Frankia sp. strains are recognized; members of each clade infect a subset of plants from among eight angiosperm families. We sequenced the genomes from three strains; their sizes varied from 5.43 Mbp for a narrow host range strain (Frankia sp. strain HFPCcI3) to 7.50 Mbp for a medium host range strain (Frankia alni strain ACN14a) to 9.04 Mbp for a broad host range strain (Frankia sp. strain EAN1pec.) This size divergence is the largest yet reported for such closely related soil bacteria (97.8%-98.9% identity of 16S rRNA genes). The extent of gene deletion, duplication, and acquisition is in concert with the biogeographic history of the symbioses and host plant speciation. Host plant isolation favored genome contraction, whereas host plant diversification favored genome expansion. The results support the idea that major genome expansions as well as reductions can occur in facultative symbiotic soil bacteria as they respond to new environments in the context of their symbioses.