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ILT2 and HLA-G expression of THP1 and THP1-G1

Upregulated and downregulated genes in CD8+ILT2+ T cells

Control of non-presence of peripheral subpopulations among the tumor infiltrate

Only some cancer patients respond to the immune-checkpoint inhibitors being used in the clinic, and other therapeutic targets are sought. Here, we investigated the HLA-G/ILT2 checkpoint in clear-cell renal-cell carcinoma (ccRCC) patients and focused on tumor-infiltrating CD8+ T lymphocytes (TIL) expressing the HLA-G receptor ILT2. Using transcriptomics and flow cytometry, we characterized both peripheral blood and tumor-infiltrating CD8+ILT2+ T cells from cancer patients as late-differentiated CD27–CD28–CD57+ cytotoxic effectors. We observed a clear dichotomy between CD8+ILT2+ and CD8+PD-1+ TIL subsets. These subsets, which were sometimes present at comparable frequencies in TIL populations, barely overlapped phenotypically and were distinguished by expression of exclusive sets of surface molecules that included checkpoint molecules and activating and inhibitory receptors. CD8+ILT2+ TILs displayed a more mature phenotype and higher expression of cytotoxic molecules. In ex vivo functional experiments with both peripheral blood T cells and TILs, CD8+ILT2+ T cells displayed significantly higher cytotoxicity and IFNγ production than their ILT2– (peripheral blood mononuclear cells, PBMC) and PD-1+ (TILs) counterparts. HLA-G expression by target cells specifically inhibited CD8+ILT2+ T-cell cytotoxicity, but not that of their CD8+ILT2– (PBMC) or CD8+PD-1+ (TIL) counterparts, an effect counteracted by blocking the HLA-G/ILT2 interaction. CD8+ILT2+ TILs may therefore constitute an untapped reservoir of fully differentiated cytotoxic T cells within the tumor microenvironment, independent of the PD1+ TILs targeted by immune therapies, and specifically inhibited by HLA-G. These results emphasize the potential of therapeutically targeting the HLA-G/ILT2 checkpoint in HLA-G+ tumors, either concomitantly with anti–PD-1/PD-L1 or in cases of nonresponsiveness to anti–PD-1/PD-L1.

Key markers of CD8+ TILs of ccRCC patients

HLA-G:ILT2 has recently been positioned as a major immune checkpoint in urologic cancers. In clear cell renal cell carcinoma (ccRCC), tumor-infiltrating CD8+ T cells expressing ILT2 are a highly cytotoxic cell population, distinct from PD1+ T cells, and whose function is inhibited by HLA-G+ targets. Here we report that ILT2 receptor can also be expressed by CD4+ T cells in urologic cancer patients. In the course of deciphering the role of these ILT2+CD4+ T cells, we found a statistical association between the tumor context and these T cells, and a positive correlation between the levels of peripheral and intra-tumoral CD4+ILT2+ T cells. Phenotypic analyses revealed that CD4+ILT2+ T cells express memory T cell (CD27−CD28−CD57+) and cytotoxicity (Tbet+Perforin+KLRG1+NKp80+GPR56+) markers, consistent with a CD4+CTL phenotype. Functional assays showed that ccRCC-infiltrating CD4+ILT2+ T cells indeed have high cytolytic properties and therefore function as proper CD4+CTLs, but are selectively inhibited by HLA-G+ targets. Clinical relevance was provided by immunohistochemical analyses on ccRCC tumor lesions with HLA-G+ HLA class II+ tumor cells next to CD4+ T cell infiltrates. Our findings provide evidence supporting that ILT2+ T cells constitute a reservoir of intratumor cytotoxic T cells that is not targeted by the current checkpoint inhibitors, but could be by anti-HLA-G/anti-ILT2 antibodies as novel immunotherapy in HLA-G+ tumors.

HLA-G was described as a molecule inhibiting NK and T cells functions through its receptor, ILT2. However, most functional studies of HLA-G were so far performed on heterogeneous immune populations and regardless of ILT2 expression. This may lead to an underestimation of the effect of HLA-G. Thus, considering the immune subpopulations sensitive to HLA-G remained an important issue in the field. Here we present a new cytometry assay to evaluate HLA-G effects on both NK and CD8+ T cell cytotoxic functions. Using flow cytometry allows for the comparison of HLA-G function on multiple subsets and multiple functions in the same time. In particular, we sharpen the analysis by specifically studying the immune subpopulations expressing HLA-G receptor ILT2. We focused our work on: IFN-gamma production and cytotoxicity (CD107a expression) by CD8+ T cells and NK cells expressing or not ILT2. We compared the expression of these markers in presence of target cells, expressing or not HLA-G1, and added a blocking antibody to reverse HLA-G inhibition. This new method allows for the discrimination of cell subsets responding and non-responding to HLA-G1 in one tube. We confirm that HLA-G-specifically inhibits the ILT2+ CD8+ T cell and ILT2+ NK cell subsets but not ILT2-negative ones. By blocking HLA-G/ILT2 interaction using an anti-ILT2 antibody we restored the cytotoxicity level, corroborating the specific inhibition of HLA-G1. We believe that our methodology enables to investigate HLA-G immune functions easily and finely towards other immune cell lineages or expressing other receptors, and might be applied in several pathological contexts, such as cancer and transplantation.

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 β2-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.

Preservation of a functional keratinocyte stem cell pool is essential to ensure the long-term maintenance of epidermis integrity, through continuous physiological renewal and regeneration in case of injury. Protecting stem cells from inflammation and immune reactions is thus a critical issue that needs to be explored. Here, we show that the immature CD49fhigh precursor cell fraction from interfollicular epidermis keratinocytes, comprising stem cells and progenitors, is able to inhibit CD4+ T-cell proliferation. Of note, both the stem cell-enriched CD49fhigh/EGFRlow subpopulation and the less immature CD49fhigh/EGFRhigh progenitors ensure this effect. Moreover, we show that HLA-G and PD-L1 immune checkpoints are overexpressed in CD49fhigh precursors, as compared to CD49flow differentiated keratinocytes. This potency may limit immune reactions against immature precursors including stem cells, and protect them from exacerbated inflammation. Further exploring this correlation between immuno-modulation and immaturity may open perspectives in allogenic cell therapies.

Only some cancer patients respond to the immune-checkpoint inhibitors being used in the clinic, and other therapeutic targets are sought. Here, we investigated the HLA-G/ILT2 checkpoint in clear-cell renal-cell carcinoma (ccRCC) patients and focused on tumor-infiltrating CD8+ T lymphocytes (TIL) expressing the HLA-G receptor ILT2. Using transcriptomics and flow cytometry, we characterized both peripheral blood and tumor-infiltrating CD8+ILT2+ T cells from cancer patients as late-differentiated CD27–CD28–CD57+ cytotoxic effectors. We observed a clear dichotomy between CD8+ILT2+ and CD8+PD-1+ TIL subsets. These subsets, which were sometimes present at comparable frequencies in TIL populations, barely overlapped phenotypically and were distinguished by expression of exclusive sets of surface molecules that included checkpoint molecules and activating and inhibitory receptors. CD8+ILT2+ TILs displayed a more mature phenotype and higher expression of cytotoxic molecules. In ex vivo functional experiments with both peripheral blood T cells and TILs, CD8+ILT2+ T cells displayed significantly higher cytotoxicity and IFNγ production than their ILT2– (peripheral blood mononuclear cells, PBMC) and PD-1+ (TILs) counterparts. HLA-G expression by target cells specifically inhibited CD8+ILT2+ T-cell cytotoxicity, but not that of their CD8+ILT2– (PBMC) or CD8+PD-1+ (TIL) counterparts, an effect counteracted by blocking the HLA-G/ILT2 interaction. CD8+ILT2+ TILs may therefore constitute an untapped reservoir of fully differentiated cytotoxic T cells within the tumor microenvironment, independent of the PD1+ TILs targeted by immune therapies, and specifically inhibited by HLA-G. These results emphasize the potential of therapeutically targeting the HLA-G/ILT2 checkpoint in HLA-G+ tumors, either concomitantly with anti–PD-1/PD-L1 or in cases of nonresponsiveness to anti–PD-1/PD-L1.

The Autoimmune Regulator (Aire) protein coordinates the negative selection of developing thymocytes by inducing the expression of hundreds of tissue‐specific antigens within the thymic medulla, which is also a primary site of the expression of the immune checkpoint HLA‐G molecule. Considering the immunomodulatory properties of Aire and HLA‐G, and considering that the role of the constitutive thymus expression of HLA‐G has not been elucidated, we studied the effect of AIRE cDNA transfection on HLA‐G expression in 4D6 thymic cells and in the HLA‐G‐positive JEG‐3 choriocarcinoma cells. Aire promoted the transactivation of HLA‐G gene by increasing the overall transcription, inducing the transcription of at least G1 and G2/G4 isoforms, and incrementing the occurrence and distribution of intracellular HLA‐G protein solely in 4D6 thymic cells. Luciferase‐based assays and chromatin immunoprecipitation experiments performed in 4D6 cells revealed that Aire targeted at least two regions within the 5′‐untranslated regulatory region (5′‐URR) extending 1·4 kb from the first ATG initiation codon. The interaction occurs independently of three putative Aire‐binding sites. These results indicate that the Aire‐induced upregulation of HLA‐G in thymic cells is likely to act through the interaction of Aire with specific HLA‐G 5′‐URR DNA‐binding factors. Such a multimeric transcriptional complex might operate in the thymus during the process of promiscuous gene expression.

In normal physiological conditions, restoration of a functional epidermal barrier is highly efficient; nevertheless, when it fails, one of the main consequences is a chronic ulcerative skin defect, one of the most frequently recognized complications of diabetes. Most of these chronic venous ulcers do not heal with conventional treatment, leading to the appearance of infections and complications in the patient. Treatments based on the use of autologous mesenchymal stem cells (MSC) have been successful; however, its implementation entails complications. The umbilical cord offers an unlimited source of adult MSC (ucMSC) from the Wharton’s jelly tissue with the same relevant features for clinical applicability and avoiding difficulties. It has recently been characterized by one specific subpopulation derived from ucMSC, the differentiated mesenchymal cells (DMCs). This subpopulation expresses the human leukocyte antigen-G (HLA-G) molecule, a strong immunosuppressive checkpoint, and vascular endothelial growth factor (VEGF), the most potent angiogenic factor. Considering the importance of developing a more effective therapy for wound treatment, especially ulcerative skin lesions, we analyzed DMC safety, efficacy, and therapeutic potential. By immunohistochemistry, umbilical cords HLA-G and VEGF positive were selected. Flow cytometry revealed that 90% of the DMC subpopulation are HLA-Gþ, CD44þ, CD73þ, CD29þ, CD105þ, CD90þ, and HLA-DR . Reverse transcription-polymerase chain reaction revealed the expression of HLA-G in all of DMC subpopulations. Upon co-culture with the DMC, peripheral blood mononuclear cell proliferation was inhibited by 50%. In a xenograft transplantation assay, DMC improved wound healing with no signs of rejection of the transplanted cells in immunocompetent mice. This study confirms that HLA-G allows allogeneic cell transplantation, and VEGF is fundamental for the restoration of the failure in blood supply. DMC population has positive effects on wound healing by promoting local angiogenesis in skin lesions. DMC could play a very important role in regenerative medicine and could be a novel allogeneic cell-therapeutic tool for wound healing., Facultad de Ciencias Médicas, Facultad de Ciencias Naturales y Museo

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 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.

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.

Made available in DSpace on 2020-12-10T20:05:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-08-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Science Without Borders Universidade de Sao Paulo-Comite Francais d'Evaluation de la Cooperation Universitaire et Scientifique avec le Bresil (USP/COFECUB) Agence Nationale de Recherches sur le Sida et les hepatites virales (ANRS) Universite Sorbonne Paris Cite (USPC) Universidade de Sao Paulo-Universite Sorbonne Paris Cite Commissariat a l'Energie Atomique et aux Energies Alternatives (CEA) Chronic hepatitis C virus (HCV) infection induces liver damage and the HCV/Human Immunodeficiency Virus (HIV)-coinfection may further contribute to its progression. The HLA-G molecule inhibits innate and adaptive immunity and may be deleterious for chronically virus-infected cells. Thus we studied 204 HCV-mono-infected patients, 142 HCV/HIV-coinfected patients, 104 HIV-mono-infected patients and 163 healthy subjects. HLA-G liver expression was similarly induced in HCV and HCV/HIV specimens, increasing with advanced fibrosis and necroinflammatory activity, and with increased levels of liver function-related enzymes. Plasma soluble HLA-G (sHLA-G) levels were higher in HCV/HIV patients compared to HCV, HIV and to healthy individuals. sHLA-G continued to be higher in coinfected patients even after stratification of samples according to degree of liver fibrosis and necroinflammatory activity when compared to mono-infected patients. Some HLA-G gene haplo-types differentiated patient groups and presented few associations with liver and plasma HLA-G expression. HLA-G thus may help to distinguish patient groups. Univ Sao Paulo, Ribeirao Preto Med Sch, Postgrad Program Basic & Appl Immunol, 3900 Bandeirantes Ave, BR-14049900 Ribeirao Preto, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Div Clin Immunol, Dept Med, 3900 Bandeirantes Ave, BR-14049900 Ribeirao Preto, Brazil Hop St Louis, Commissariat Energie Atom & Energies Alternat, Direct Rech Fondamentale, Inst Biol Francois Jacob,Serv Rech Hematoimmunol, 1 Ave Claude Vellefaux, F-75475 Paris, France Univ Paris Diderot, Inst Rech St Louis, HIPI, UMR 976, 1 Ave Claude Vellefaux, F-75475 Paris, France Univ Sao Paulo, Ribeirao Preto Med Sch, Postgrad Program Genet, 3900 Bandeirantes Ave, BR-14049900 Ribeirao Preto, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Div Gastroenterol, Dept Med, 3900 Bandeirantes Ave, BR-14049900 Ribeirao Preto, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Div Infect & Trop Dis, Dept Med, 3900 Bandeirantes Ave, BR-14049900 Ribeirao Preto, Brazil Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Pathol, 3900 Bandeirantes Ave, BR-14049900 Ribeirao Preto, Brazil Sao Paulo State Univ, Dept Pathol, Sch Med, Av Dr Mario Rubens Guimaraes Montenegro, BR-18618687 Botucatu, SP, Brazil Univ Sao Paulo, Fac Filosofia Ciencias & Letras Ribeirao Preto, Dept Quim, 3900 Bandeirantes Ave, BR-14049900 Ribeirao Preto, Brazil Sao Paulo State Univ, Dept Pathol, Sch Med, Av Dr Mario Rubens Guimaraes Montenegro, BR-18618687 Botucatu, SP, Brazil CAPES: 653/09 Science Without Borders: 236754/2012-2 CNPq: 466036/2013-5 CNPq: 304931/2014-4 Universidade de Sao Paulo-Comite Francais d'Evaluation de la Cooperation Universitaire et Scientifique avec le Bresil (USP/COFECUB): 2017.1.1337.1.5 Agence Nationale de Recherches sur le Sida et les hepatites virales (ANRS): 12296/2013 : 406594/2013-9 : 401641/2013-9

International audience; 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.

The HLA-G 5’URR extending 1.4 kb from the ATG presents a unique set of regulatory elements among HLA genes. Several variable sites have been described that coincide with or are close to these elements, thus HLA-G 5′URR polymorphism might influence the HLA-G expression level. We cloned the ten most frequent HLA-G 5′URR haplotypes to evaluate their activity on a luciferase reporter gene in HLA-G+ cell lines (JEG-3/choriocarcinoma and FON+/melanoma). We also investigated associations between the plasma HLA-G (sHLA-G) levels and the HLA-G 5′URR variability in 157 healthy individuals. Cell lines were transfected with pGL3-Basic vector constructions containing HLA-G 5′URR sequences. The G010101a (in JEG-3) and G010101b (in FON+) haplotypes exhibited higher promoter activity, whereas the G010101d (in JEG-3) and G010102a (in FON+) haplotypes exhibited lower promoter activity. In the presence of HLA-G inducers (interferon-β and progesterone) or repressors (cyclopamine) HLA-G promoter activity was modulated, but certain haplotypes exhibited differential responses. No strict association was observed between plasma sHLA-G levels and the 5′URR haplotypes or genotypes; however, the G010101b haplotype was underrepresented among HLA-G-negative plasmas. Therefore, the HLA-G 5′URR polymorphism may have an impact on the modulation of HLA-G gene expression, but alone provides a limited predictive value for sHLA-G levels in vivo.

Bronchiolitis obliterans syndrome (BOS) after lung transplantation (LTx) results from bronchial epithelial cell (BECs) damages, thought to be orchestrated by T cells primed by antigen-presenting cell presenting alloantigens. In this cell cross-talk, BECs are also suspected to play a pivotal immunosuppressive role in T cell alloreactivity. We studied the immunomodulating role of BECs in a human ex vivo model of allogeneic T cell response, both in healthy subjects and LTx recipients.BECs from 35 LTx recipients (n = 22 stable, n = 13 BOS) and healthy controls (n = 25) were cultured as primary cell cultures. Their inhibitory capacities through the involvement of tolerogenic molecules (HLA-G, TGF-β, and IL-10) were tested on a mixed lymphocyte reaction between antigen-presenting cells and recipient T cells.Control BECs inhibited T cell alloproliferation by a mean of 53 ± 7%. This inhibitory effect of BECs was significantly reduced in the stable LTx group (24 ± 8%, P = 0.009), but not in the BOS TxP group (53 ± 10%, P = 0.97). Neutralization of HLA-G, TGF-β, and IL-10 partially restored T cell alloproliferation, arguing for their involvement in the immunosuppressive effect of BECs. BECs culture supernatant from stable LTx patients with impaired BEC properties showed a skewed Th2-type secretion profile (high IL-4/IFN-γ ratio).The inhibitory properties of BECs are dysregulated in stable LTx recipients, which could suggest their instrumental role in the initiation of BOS process and potential targeted therapies.

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.

Introduction: HLA-G plays a key role on immune tolerance. Pathogens can induce soluble HLA-G (sHLA-G) production to down-regulate the host immune response, creating a tolerogenic environment favorable for their dissemination. To our knowledge, no study has yet been conducted to assess the relationship between sHLA-G and geohelminth infections. Methods: The study was conducted in Allada, Southeastern Benin, from 2011 - 2014. The study population encompassed 400 pregnant women, included before the end of the 28th week of gestation and followed-up until delivery. At two antenatal care visits and at delivery, stool and blood samples were collected. Helminths were diagnosed by means of the Kato-Katz concentration technique. We used quantile regression to analyze the association between helminth infections and sHLA-G levels during pregnancy. Results: sHLA-G levels gradually increased during pregnancy and reached maximal levels at delivery. Prevalence of helminth infections was low, with a majority of hookworm infections. We found significantly more hookworm-infected women above the 80th quantile (Q80) of the distribution of the mean sHLA-G level (p < 0.03, multivariate quantile regression). Considering only women above the Q80 percentile, the mean sHLA-G level was significantly higher in hookworm-infected compared to uninfected women (p = 0.04). Conclusion: High levels of sHLA-G were associated with hookworm infection in pregnant women. This result is consistent with the potential involvement of sHLA-G in immune tolerance induced by helminths during pregnancy.

International audience; 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.

// Layale Yaghi 1, 2, 3 , Isabelle Poras 1, 2 , Renata T. Simoes 1, 2, 4 , Eduardo A. Donadi 5 , Jorg Tost 6, 7 , Antoine Daunay 6 , Bibiana Sgorla de Almeida 1, 2, 5 , Edgardo D. Carosella 1, 2 , Philippe Moreau 1, 2 1 Commissariat a l’Energie Atomique et aux Energies Alternatives, Institut des Maladies Emergentes et des Therapies Innovantes, Service de Recherches en Hemato-Immunologie, Hopital Saint-Louis, Paris, France 2 Universite Paris-Diderot, Sorbonne Paris-Cite, UMR E5, Institut Universitaire d’Hematologie, Hopital Saint-Louis, Paris, France 3 Lebanese University, School of Medicine, Hadath, Lebanon 4 Instituto de Ensino e Pesquisa da Santa Casa de Belo Horizonte, IEP/SCBH, Belo Horizonte, Minas Gerais, Brasil 5 Divisao de Imunologia Clinica, Departamento de Clinica Medica, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Sao Paulo, Brasil 6 Centre d’Etude du Polymorphisme Humain, Fondation Jean-Dausset, Laboratory for Functional Genomics, Paris, France 7 Commissariat a l’Energie Atomique et aux Energies Alternatives, Centre National de Genotypage, Laboratory for Epigenetics and Environment, Evry, France Correspondence to: Philippe Moreau, email: philippe.moreau@cea.fr Keywords: HLA-G, HIF-1, exon 2 HRE, glioma Received: July 02, 2016 Accepted: August 20, 2016 Published: August 26, 2016 ABSTRACT HLA-G is an immune checkpoint molecule with specific relevance in cancer immunotherapy. It was first identified in cytotrophoblasts, protecting the fetus from maternal rejection. HLA-G tissue expression is very restricted but induced in numerous malignant tumors such as glioblastoma, contributing to their immune escape. Hypoxia occurs during placenta and tumor development and was shown to activate HLA-G . We aimed to elucidate the mechanisms of HLA-G activation under conditions combining hypoxia-mimicking treatment and 5-aza-2’deoxycytidine, a DNA demethylating agent used in anti-cancer therapy which also induces HLA-G . Both treatments enhanced the amount of HLA-G mRNA and protein in HLA-G negative U251MG glioma cells. Electrophoretic Mobility Shift Assays and luciferase reporter gene assays revealed that HLA-G upregulation depends on Hypoxia Inducible Factor-1 (HIF-1) and a hypoxia responsive element (HRE) located in exon 2. A polymorphic HRE at –966 bp in the 5’UT region may modulate the magnitude of the response mediated by the exon 2 HRE. We suggest that therapeutic strategies should take into account that HLA-G expression in response to hypoxic tumor environment is dependent on HLA-G gene polymorphism and DNA methylation state at the HLA-G locus.

Only some cancer patients respond to the immune-checkpoint inhibitors being used in the clinic, and other therapeutic targets are sought. Here, we investigated the HLA-G/ILT2 checkpoint in clear-cell renal-cell carcinoma (ccRCC) patients and focused on tumor-infiltrating CD8

The study aims to determine if genetic polymorphisms in the human leukocyte antigen (HLA)-G gene are associated with age-related macular degeneration (AMD). HLA-G is important for immunological tolerance, and it is also known to have angiogenic effects. Polymorphisms in the 5'-upstream regulatory region (URR) and 3'-untranslated region (UTR) of HLA-G have been associated with a number of diseases, especially with respect to a 14 bp insertion/deletion (ins/del) polymorphism in the 3'UTR. Full gene sequencing was performed on a cohort of 146 AMD patients and 63 healthy controls aged 60 years or older and HLA-G haplotypes were determined. Analyses were performed on a publicly available gene expression dataset from the NCBI GEO database (accession number GSE29801) from which expression data for HLA-G, -C and -A were extracted. Analysis of the GEO dataset showed that both HLA-G and -C was expressed in the back of the eye and that expression was upregulated in the macular area of AMD. No differences were observed between patients and controls when analysing the distribution of haplotypes in the HLA-G promoter, coding region, 3'UTR or the 14 bp ins/del polymorphism of the 3'UTR. The increased expression of HLA-G in the macula of AMD patients indicates a role of HLA-G in the micro environment as part of the AMD pathogenesis. This is supported by the expression of HLA-C, which has previously been shown to play a role in AMD. The HLA-G haplotype distribution did not display any differences between AMD patients and controls. This article is protected by copyright. All rights reserved.

// Danli Wu 1 , Isere Kuiaste 2 , Philippe Moreau 3,4 , Edgardo Carosella 3,4 and Patricia Yotnda 1 1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston Methodist Hospital, Texas Children Hospital, Houston, TX, USA 2 Breast Cancer Center, Baylor College of Medicine, Houston, TX, USA 3 CEA, Institut des Maladies Emergentes et des Therapies Innovantes (IMETI), Service de Recherches en Hemato-Immunologie (SRHI), Hopital Saint-Louis, Paris, France 4 Universite Paris-Diderot, Sorbonne Paris-Cite, UMR E5, Institut Universitaire d’Hematologie, Hopital Saint-Louis, Paris, France Correspondence to: Patricia Yotnda, email: // Keywords : cancers, immunosuppression, HLA-G, ILT2, lymphocytes Received : June 13, 2015 Accepted : September 17, 2015 Published : October 09, 2015 Abstract Several studies have demonstrated that the antitumor activities of both T and natural killer (NK) effector populations are limited by the immunosuppressive strategies of tumors. In several malignant transformations, the expression of HLA-G by tumor cells rises dramatically, rendering them strongly immunosuppressive. In this study, we postulated that the absence of HLA-G receptors would prevent the immunosuppressive effects of both soluble and membrane-bound HLA-G. Thus, we investigated the therapeutic potential of effector NK cells genetically modified to downregulate the expression of ILT2 (HLA-G receptor) on their cell surfaces. We have shown that the proliferation of modified NK is still dependent on stimulation signals (no malignant transformation). ILT2 - NK cells proliferate, migrate, and eliminate HLA-G negative targets cells to the same extent parental NK cells do. However, in the presence of HLA-G positive tumors, ILT2 - NK cells exhibit superior proliferation, conjugate formation, degranulation, and killing activities compared to parent NK cells. We tested the effectiveness of ILT2 - NK cells in vivo using a xenograft cancer model and found that silencing ILT2 rescued their anti-tumor activity. We believe that combining ILT2 - NK cells with existing therapeutic strategies will strengthen the antitumor response in cancer patients.

Context Overexpression of immune checkpoint molecules affects tumor-specific T-cell immunity in the cancer microenvironment, and can reshape tumor progression and metastasis. Antibodies targeting checkpoints could restore antitumor immunity by blocking the inhibitory receptor-ligand interaction. Objective To analyze data and current trends in immune checkpoint targeting therapy for urologic cancers. Evidence acquisition Systematic literature search for clinical trials in the PubMed and Cochrane databases up to August 2014 according to Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Endpoints included oncologic results, tumor response rates, safety, and tolerability. Evidence synthesis Anti-CTLA-4 monotherapy has demonstrated biochemical responses in prostate cancer. One phase 3 trial assessing ipilimumab efficacy in castration-resistant disease was negative overall. Nevertheless, ipilimumab may significantly improve overall survival compared with placebo in subgroups of patients with favorable prognostic features. In renal cancer, phase 1 trials showed interesting stabilization or long-lasting objective response rates approaching 50% using anti-PD-1/PD-L1 drugs in heavily pretreated metastatic patients. In bladder cancer, one phase 2 trial indicated a good safety profile for ipilimumab as a neoadjuvant drug before radical cystectomy. Overall, immune-related effects such as colitis and dermatitis were common and well tolerated. Conclusions Our systematic review shows that antibodies blocking immune checkpoints offer interesting and long-lasting response rates in heavily pretreated patients with advanced urologic cancers. More promising results are currently provided by anti-CTLA-4 antibodies in prostate cancer and by PD-1/PD-L1 inhibitors in renal cancer. These should encourage new clinical trials of immune therapy combinations and immunotherapy monotherapy combined with conventional anticancer drugs. In bladder cancer, the use of targeted immunotherapy still remains underevaluated; however, preliminary results reported at recent conferences seem encouraging. Patient summary Data from studies support the activity and safety of immune checkpoint inhibitors in urologic cancers, alone or in combination with conventional cancer therapies. Encouraging data in other oncologic fields could translate into interesting responses in urological cancers.

// Francois Desgrandchamps 1, 2, * , Joel LeMaoult 1, 3, * , Annabelle Goujon 1, 2 , Adrien Riviere 1, 2 , Antonio Rivero-Juarez 1, 4 , Malika Djouadou 1, 2 , Amory de Gouvello 1, 2 , Clement Dumont 1, 5 , Ching-Lien Wu 1, 3 , Stephane Culine 1, 5 , Jerome Verine 1, 6 , Nathalie Rouas-Freiss 1, 3 , Christophe Hennequin 1, 7 , Alexandra Masson-Lecomte 1, 2 and Edgardo D. Carosella 1, 3 1 CEA, DRF-Francois Jacob Institute, Research Division in Hematology and Immunology (SRHI), Saint-Louis Hospital, Paris, France 2 AP-HP, Saint-Louis Hospital, Department of Urology, Paris, France 3 University Paris Diderot, Sorbonne Paris Cite, UMR E_5 Institut Universitaire d’Hematologie, Saint-Louis Hospital, Paris, France 4 Infectious Diseases Unit, Instituto Maimonides de Investigacion Biomedica de Cordoba (IMIBIC), Hospital Universitario Reina Sofia, Universidad de Cordoba, Cordoba, Spain 5 AP-HP, Saint-Louis Hospital, Department of Medical Oncology, Paris, France 6 AP-HP, Saint-Louis Hospital, Department of Pathology, Paris, France 7 AP-HP, Saint-Louis Hospital, Department of Radiotherapy, Paris, France * These authors contributed equally to this work Correspondence to: Joel LeMaoult, email: Joel.LeMaoult@cea.fr Keywords: ILT2; HLA-G; immune checkpoint; bladder; cancer Received: January 20, 2018 Accepted: August 03, 2018 Published: September 04, 2018 ABSTRACT Background and Objective: Recurrence of non-muscle invasive bladder cancer (NMIBC) after initial management occurs in 60–70% of patients. Predictive criteria for recurrence remain only clinical and pathological. The aim of this study was to investigate the prognostic significance of the proportion of checkpoint HLA-G’s receptor ILT2-expressing peripheral CD8 + T cells. Results: The proportion of CD4 + ILT2 + and CD8 + ILT2 + T cells was not increased in NMIBC compared to controls. However, a strong association was found between recurrence and CD8 + ILT2 + T cell population levels ( p = 0.0006). Two-year recurrence-free survival was 83% in patients with less than 18% CD8 + ILT2 + T cells, 39% in the intermediary group, and 12% in patients with more than 46% CD8 + ILT2 + T cells. Multivariate analyses demonstrated that the proportion of CD8 + ILT2 + T cells was an independent predictive factor for recurrence. Adding CD8 + ILT2 + T cells population level to clinical variables increased the predictive accuracy of the model by 4.5%. Materials and Methods: All patients treated for NMIBC between 2012 and 2014 were included prospectively. Blood samples, tumor and clinico-pathological characteristics were collected. HLA-G expression was measured using IHC, and CD8 + ILT2 + T cell levels using flow cytometry. Association between HLA-G and CD8 + ILT2 + T cell population levels with NMIBC risk of recurrence was investigated using Cox regression analyses. Prediction was measured using the concordance index statistic. Conclusions: We demonstrated a strong association between the proportion of circulating CD8 + ILT2 + T cells and NMIBC risk of recurrence. Gain in prediction was substantial. If externally validated, such immunological marker could be integrated to predict NMIBC recurrence.

Volume 83, no. 10, p. 3918–3925, 2015, [https://doi.org/10.1128/IAI.00803-15][1]. Page 3922: Corrected panels for Fig. 3e and f should appear as shown below. The panels with possible duplication were the 0′ points for THP1 cells. This does not affect the results, since the 0′ point was added

The HLA-G 5’URR extending 1.4 kb from the ATG presents a unique set of regulatory elements among HLA genes. Several variable sites have been described that coincide with or are close to these elements, thus HLA-G 5′URR polymorphism might influence the HLA-G expression level. We cloned the ten most frequent HLA-G 5′URR haplotypes to evaluate their activity on a luciferase reporter gene in HLA-G+ cell lines (JEG-3/choriocarcinoma and FON+/melanoma). We also investigated associations between the plasma HLA-G (sHLA-G) levels and the HLA-G 5′URR variability in 157 healthy individuals. Cell lines were transfected with pGL3-Basic vector constructions containing HLA-G 5′URR sequences. The G010101a (in JEG-3) and G010101b (in FON+) haplotypes exhibited higher promoter activity, whereas the G010101d (in JEG-3) and G010102a (in FON+) haplotypes exhibited lower promoter activity. In the presence of HLA-G inducers (interferon-β and progesterone) or repressors (cyclopamine) HLA-G promoter activity was modulated, but certain haplotypes exhibited differential responses. No strict association was observed between plasma sHLA-G levels and the 5′URR haplotypes or genotypes; however, the G010101b haplotype was underrepresented among HLA-G-negative plasmas. Therefore, the HLA-G 5′URR polymorphism may have an impact on the modulation of HLA-G gene expression, but alone provides a limited predictive value for sHLA-G levels in vivo.

// Valli De Re 1, * , Laura Caggiari 1, * , Lara Mussolin 2, ** , Emanuele Stefano d’Amore 3, ** , Barbara Famengo 3, ** , Mariangela De Zorzi 1 , Lia Martina 1 , Caterina Elia 4 , Marta Pillon 5, ** , Nicola Santoro 6, ** , Paola Muggeo 6, ** , Salvatore Buffardi 7, ** , Maurizio Bianchi 8, ** , Alessandra Sala 9, ** , Piero Farruggia 10, ** , Luciana Vinti 11, ** , Edgardo D. Carosella 12 , Roberta Burnelli 13, ** and Maurizio Mascarin 4, ** 1 Immunopathology and Cancer Biomarkers, Department of Translational Research, CRO Aviano National Cancer Institute, Aviano, Italy 2 Clinic of Pediatric Hemato-Oncology, Department of Women’s and Children’s Health, University of Padova, Institute of Paediatric Research Fondazione Citta della Speranza, Padova, Italy 3 Department of Pathology, San Bortolo Hospital, Vicenza, Italy 4 Pediatric Radiotherapy Unit, CRO Aviano National Cancer Institute, Aviano, Italy 5 Clinic of Paediatric Hemato-Oncology, Department of Women’s and Children’s Health, University of Padova, Padova, Italy 6 Department of Paediatric Hemato-Oncology, University of Bari, Bari, Italy 7 Department of Paediatric Hemato-Oncology, Santobono-Pausilipon Children’s Hospital, Napoli, Italy 8 Department of Paediatric Hemato-Oncology, Regina Margherita Children’s Hospital, Torino, Italy 9 Department of Paediatrics, Ospedale San Gerardo, University of Milano-Bicocca, Fondazione MBBM, Monza, Italy 10 Pediatric Hematology and Oncology Unit, Oncology Department, A.R.N.A.S. Ospedali Civico, Di Cristina e Benfratelli, Palermo, Italy 11 Department of Paediatric Hemato-Oncology, IRCCS Ospedale Bambino Gesu, Roma, Italy 12 Department of Hemato-Immunological Research, Institut des Maladies Emergentes et des Therapies Innovantes (iMETI), Hopital Saint Louis, Paris, France 13 Pediatric Hematology-Oncology, Azienda Ospedaliera Universitaria, Ospedale Sant’Anna, Ferrara, Italy * These authors have contributed equally to this work ** These authors are members of the AIEOP group Correspondence to: Valli De Re, email: vallidere@gmail.com Keywords: pediatric hodgkin lymphoma; HLA-G; event-free survival; 3’UTR polymorphism; +3027 C/A genotype Received: July 21, 2017 Accepted: October 28, 2017 Published: November 18, 2017 ABSTRACT In this study, we tested whether polymorphisms in human leukocyte antigen G (HLA-G) were associated with event-free survival (EFS) in pediatric Hodgkin’s lymphoma (HL). We evaluated the association of HLA-G 3’-UTR polymorphisms with EFS in 113 pediatric HL patients treated using the AIEOP LH-2004 protocol. Patients with the +3027-C/A genotype (rs17179101, UTR-7 haplotype) showed lower EFS than those with the +3027-C/C genotype (HR= 3.23, 95%CI: 0.99-10.54, P=0.012). Female patients and systemic B symptomatic patients with the HLA-G +3027 polymorphism showed lower EFS. Multivariate analysis showed that the +3027-A polymorphism (HR 3.17, 95%CI 1.16-8.66, P=0.025) was an independent prognostic factor. Immunohistochemical analysis showed that HL cells from patients with the +3027-C/A genotype did not express HLA-G. Moreover, HLA-G +3027 polymorphism improved EFS prediction when added to the algorithm for therapeutic group classification of pediatric HL patients. Our findings suggest HLA-G +3027 polymorphism is a prognostic marker in pediatric HL patients undergoing treatment according to LH-2004 protocol.

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.