ABSTRACT
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.
Subject(s)
Carcinoma, Renal Cell , Kidney Neoplasms , Cell Membrane/metabolism , HLA-G Antigens/chemistry , HLA-G Antigens/genetics , Humans , Protein Isoforms/genetics , Protein Isoforms/metabolismABSTRACT
Mesenchymal stem cells (MSCs) are isolated from multiple biological tissues-adult bone marrow and adipose tissues and neonatal tissues such as umbilical cord and placenta. In vitro, MSCs show biological features of extensive proliferation ability and multipotency. Moreover, MSCs have trophic, homing/migration and immunosuppression functions that have been demonstrated both in vitro and in vivo. A number of clinical trials are using MSCs for therapeutic interventions in severe degenerative and/or inflammatory diseases, including Crohn's disease and graft-versus-host disease, alone or in combination with other drugs. MSCs are promising for therapeutic applications given the ease in obtaining them, their genetic stability, their poor immunogenicity and their curative properties for tissue repair and immunomodulation. The success of MSC therapy in degenerative and/or inflammatory diseases might depend on the robustness of the biological functions of MSCs, which should be linked to their therapeutic potency. Here, we outline the fundamental and advanced concepts of MSC biological features and underline the biological functions of MSCs in their basic and translational aspects in therapy for degenerative and/or inflammatory diseases.
Subject(s)
Mesenchymal Stem Cells/metabolism , Adipose Tissue/cytology , Bone Marrow Cells/cytology , Cell Differentiation , Cell- and Tissue-Based Therapy , Graft vs Host Disease/prevention & control , Humans , Immunosuppression Therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Wnt Signaling PathwayABSTRACT
BACKGROUND: Human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense can be diagnosed in the early hemolymphatic stage (stage 1 [S1]) or meningoencephalitic stage (stage 2 [S2]). Importantly, individuals harbouring high and specific antibody responses to Tbg antigens but negative parasitology are also diagnosed in the field (seropositive [SERO]). Whereas some develop the disease in the months following their initial diagnosis (SERO/HAT), others remain parasitologically negative for long periods (SERO) and are apparently able to control infection. Human leucocyte antigen (HLA)-G, an immunosuppressive molecule, could play a critical role in this variability of progression between infection and disease. METHODS: Soluble HLA-G (sHLA-G) was measured in plasma for patients in the SERO (n = 65), SERO/HAT (n = 14), or HAT (n = 268) group and in cerebrospinal fluid for patients in S1 (n = 55), early S2 (n = 93), or late S2 (n = 110). Associations between these different statuses and the soluble level or genetic polymorphisms of HLA-G were explored. RESULTS: Plasma sHLA-G levels were significantly higher in HAT (P = 6 × 10-7) and SERO/HAT (P = .007) than SERO patients. No difference was observed between the SERO/HAT and HAT groups. Within the HAT group, specific haplotypes (HG010102 and HG0103) displayed increased frequencies in S1 (P = .013) and late S2 (P = .036), respectively. CONCLUSIONS: These results strongly suggest the involvement of HLA-G in HAT disease progression. Importantly, high plasma sHLA-G levels in SERO patients could be predictive of subsequent disease development and could represent a serological marker to help guide therapeutic decision making. Further studies are necessary to assess the predictive nature of HLA-G and to estimate both sensitivity and specificity.
Subject(s)
HLA-G Antigens/blood , Trypanosomiasis, African/blood , Adult , Biomarkers/blood , Disease Progression , Female , Haplotypes , Humans , Male , Prognosis , Trypanosoma brucei gambiense , Trypanosomiasis, African/physiopathology , Trypanosomiasis, African/prevention & controlABSTRACT
Inhibition of B cells constitutes a rational approach for treating B cell-mediated disorders. We demonstrate in this article that the engagement of the surface Ig-like transcript 2 (ILT2) inhibitory receptor with its preferential ligand HLA-G is critical to inhibit B cell functions. Indeed, ILT2-HLA-G interaction impedes both naive and memory B cell functions in vitro and in vivo. Particularly, HLA-G inhibits B cell proliferation, differentiation, and Ig secretion in both T cell-dependent and -independent models of B cell activation. HLA-G mediates phenotypic and functional downregulation of CXCR4 and CXCR5 chemokine receptors on germinal center B cells. In-depth analysis of the molecular mechanisms mediated by ILT2-HLA-G interaction showed a G0/G1 cell cycle arrest through dephosphorylation of AKT, GSK-3ß, c-Raf, and Foxo proteins. Crucially, we provide in vivo evidence that HLA-G acts as a negative B cell regulator in modulating B cell Ab secretion in a xenograft mouse model. This B cell regulatory mechanism involving ILT2-HLA-G interaction brings important insight to design future B cell-targeted therapies aimed at reducing inappropriate immune reaction in allotransplantation and autoimmune diseases.
Subject(s)
Antigens, CD/immunology , B-Lymphocytes/immunology , HLA-G Antigens/immunology , Lymphocyte Activation/immunology , Receptors, Immunologic/immunology , Animals , Cell Differentiation/immunology , Cell Movement/immunology , Cell Proliferation , Cells, Cultured , Down-Regulation , Female , Forkhead Transcription Factors/metabolism , G1 Phase Cell Cycle Checkpoints/immunology , Germinal Center/immunology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Immunologic Memory/immunology , Leukocyte Immunoglobulin-like Receptor B1 , Mice , Mice, Inbred BALB C , Palatine Tonsil/immunology , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Receptors, CXCR4/biosynthesis , Receptors, CXCR5/biosynthesis , Th2 Cells/immunology , Transplantation, HeterologousABSTRACT
Human leukocyte antigen-G (HLA-G) expression by tumors has been evidenced in numerous malignancies in association with poor prognosis and resistance to immunotherapy in humans. Particularly, soluble form of HLA-G was measured at high concentrations in malignant effusions and plasma from cancer patients, and inhibits antitumor immune cells in vitro through interaction with immunoglobulin-like transcript (ILT) receptors. Nevertheless, in vivo study demonstrating that HLA-G secretion by tumor cells allows their escape from immunosurveillance remained to be established. Despite nondescribed murine homolog, direct functional interaction of HLA-G with murine paired immunoglobulin-like receptor (PIR)-B, ortholog of human ILT receptors, enables to investigate its role in vivo. Immunocompetent mice were injected either with syngeneic tumor cells co-expressing HLA-G5, the main soluble HLA-G isoform, and the conformation stabilizer human ß2-microglubulin (hß2m), or with hß2m+ HLA-G5- tumor cells. hß2m expressed at both tumor cell surface acted as a tumor antigen triggering a specific humoral response. Interestingly, although hß2m+ HLA-G5- tumors were rejected, secreted HLA-G5 provided hß2m+ HLA-G5+ tumors a protection against hß2m-elicited immune rejection, enabling such immunogenic tumors to grow similarly to a poorly immunogenic tumor. HLA-G5 tumor expression was associated with local and peripheral immunosuppression, characterized by dampened anti-hß2m B-cell response, quantitative and functional T-and B-cell defects, accumulation of myeloid-derived suppressor cells able to inhibit T-cell proliferation and reduced T- and B-cell tumor infiltrate. Our study provides the first in vivo proof that soluble HLA-G counteracts tumor rejection and reinforces the importance to consider HLA-G as a promising target to optimize current cancer immunotherapies.
Subject(s)
B-Lymphocytes/immunology , Breast Neoplasms/immunology , HLA-G Antigens/immunology , Melanoma/immunology , Monitoring, Immunologic , T-Lymphocytes/immunology , Tumor Escape/immunology , Animals , Antigens, Neoplasm , Apoptosis , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , HLA-G Antigens/metabolism , Humans , Immunoenzyme Techniques , Immunoprecipitation , Lymphocyte Activation , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, Inbred BALB C , T-Lymphocytes/metabolism , T-Lymphocytes/pathology , Tumor Cells, CulturedABSTRACT
Both human leukocyte antigen G (HLA-G) and multipotential mesenchymal stem/stromal cells (MSCs) exhibit immunomodulatory functions. In allogeneic tranplantation, the risks of acute and chronic rejection are still high despite improvement in immunosuppressive treatments, and the induction of a state of tolerance to alloantigens is not achieved. Immunomodulatory properties of MSCs and HLA-G in human allogeneic tranplantation to induce tolerance appears attractive and promising. Interestingly, we and others have demonstrated that MSCs can express HLA-G. In this review, we focus on the expression of HLA-G by MSCs and discuss how to ensure and improve the immunomodulatory properties of MSCs by selectively targeting MSCs expressing HLA-G (MSCs(HLA-G+)). We also discuss the possible uses of MSCs(HLA-G+) for therapeutic purposes, notably, to overcome acute and chronic immune rejection in solid-organ allogeneic transplantation in humans. Since MSCs are phenotypically and functionally heterogeneous, it is of primary interest to have specific markers ensuring that they have strong immunosuppressive potential and HLA-G may be a valuable candidate.
Subject(s)
Biological Therapy/methods , HLA-G Antigens/immunology , Immunosuppression Therapy/methods , Mesenchymal Stem Cells/immunology , Animals , HLA-G Antigens/biosynthesis , Humans , Immunosuppressive Agents/immunology , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/metabolism , Transplantation Immunology , Transplantation, Homologous/methodsABSTRACT
Bone-marrow mesenchymal stem cells (MSCs) are the origin of bone-forming cells with immunomodulation potential. HLA-G5 is among the generated immunosuppressive molecules. HLA-G proteins play a crucial role in promoting the acceptance of allografts. However, the mechanisms regulating the expression of HLA-G5 in human MSCs are unknown. We induced differentiation of MSCs and found that HLA-G5 was greatly up-regulated only in osteoblastic cells (+63% for mRNA). Growth plates and bone callus postfracture in adults showed that only bone-lining cells and mesenchymal progenitors were positive for HLA-G5. Use of gene silencing and dominant-negative factors revealed that HLA-G5 depends on the expression and function of the skeletogenesis master genes RUNX2 and DLX5. In addition, HLA-G5 could directly inhibit osteoclastogenesis by acting on monocytes through SHP1. However, in mature osteoblasts, the expression of HLA-G5 protein was greatly suppressed whereas the proosteoclastogenic factor, RANKL, was concomitantly increased. Down-regulation of HLA-G5 expression during the maturation of osteoblasts was due to binding of the repressor GLI3, a signal transducer of the Hedgehog pathway, to the GLI binding element within the HLA-G promoter. Our findings show that mesenchymal progenitors and osteoblastic cells specifically express HLA-G5 during osteogenesis, with a key role in bone homeostasis.
Subject(s)
Bone and Bones/metabolism , HLA-G Antigens/genetics , Mesenchymal Stem Cells/metabolism , Osteoblasts/metabolism , Osteogenesis/genetics , Adult , Bone and Bones/cytology , Cell Line, Tumor , Cell Lineage/genetics , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Gene Expression Regulation , HLA-G Antigens/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Homeostasis/genetics , Humans , Immunohistochemistry , Microscopy, Fluorescence , Models, Genetic , Osteoblasts/cytology , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
BACKGROUND: The immunosuppressive properties of HLA-G protein can create a tolerogenic environment that may allow Plasmodium falciparum to avoid host immune responses. There are known associations between high levels of circulating soluble HLA-G (sHLA-G) and either parasite or viral infections and it has been suggested that the induction of sHLA-G expression could be a mechanism via which infectious agents subvert host immune defence. The study presented here is the first to investigate the possible association between sHLA-G and malaria or malaria related risk factors in Benin. METHODS: A parasitological and clinical follow-up of 165 mothers and their newborns from delivery through to one year of age was conducted in the Tori Bossito area of southern Benin. Plasma levels of sHLA-G were determined by ELISA in maternal peripheral and cord blood and again in infants' peripheral blood at 3, 6, 9 and 12 months of age. The associations between the levels of sHLA-G and malaria risk factors were investigated through multivariate mixed models. RESULTS: Strong correlations were observed between the maternal and cord plasma concentrations of sHLA-G. In multivariate analyses, high cord plasma levels of sHLA-G were independently associated with (i) low birth weight and (ii) an increased risk of P. falciparum infection in infancy. CONCLUSION: These results show for the first time the possible involvement of sHLA-G in generating immune tolerance during pregnancy-associated malaria. Soluble HLA-G may represent a useful marker of susceptibility to malaria in infants and be associated with the higher susceptibility to infection observed for LBW children.
Subject(s)
Disease Susceptibility , HLA-G Antigens/blood , Infant, Low Birth Weight , Malaria, Falciparum/epidemiology , Adolescent , Adult , Benin/epidemiology , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Female , Humans , Infant , Infant, Newborn , Male , Middle Aged , Pregnancy , Risk Assessment , Young AdultABSTRACT
Preeclampsia is a syndrome with multiple etiologies. The diagnosis can be made without proteinuria in the presence of dysfunction of at least 1 organ associated with hypertension. The common pathophysiological pathway includes endothelial cell activation, intravascular inflammation, and syncytiotrophoblast stress. There is evidence to support, among others, immunologic causes of preeclampsia. Unlike defense immunology, reproductive immunology is not based on immunologic recognition systems of self/non-self and missing-self but on immunotolerance and maternal-fetal cellular interactions. The main mechanisms of immune escape from fetal to maternal immunity at the maternal-fetal interface are a reduction in the expression of major histocompatibility complex molecules by trophoblast cells, the presence of complement regulators, increased production of indoleamine 2,3-dioxygenase, activation of regulatory T cells, and an increase in immune checkpoints. These immune protections are more similar to the immune responses observed in tumor biology than in allograft biology. The role of immune and nonimmune decidual cells is critical for the regulation of trophoblast invasion and vascular remodeling of the uterine spiral arteries. Regulatory T cells have been found to play an important role in suppressing the effectiveness of other T cells and contributing to local immunotolerance. Decidual natural killer cells have a cytokine profile that is favored by the presence of HLA-G and HLA-E and contributes to vascular remodeling. Studies on the evolution of mammals show that HLA-E, HLA-G, and HLA-C1/C2, which are expressed by trophoblasts and their cognate receptors on decidual natural killer cells, are necessary for the development of a hemochorial placenta with vascular remodeling. The activation or inhibition of decidual natural killer cells depends on the different possible combinations between killer cell immunoglobulin-like receptors, expressed by uterine natural killer cells, and the HLA-C1/C2 antigens, expressed by trophoblasts. Polarization of decidual macrophages in phenotype 2 and decidualization of stromal cells are also essential for high-quality vascular remodeling. Knowledge of the various immunologic mechanisms required for adequate vascular remodeling and their dysfunction in case of preeclampsia opens new avenues of research to identify novel biological markers or therapeutic targets to predict or prevent the onset of preeclampsia.
ABSTRACT
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.
Subject(s)
HLA-G Antigens/chemistry , Neoplasms/immunology , Protein Multimerization , Tumor Microenvironment , Cell Line, Tumor , Humans , Interferon-beta/pharmacology , Interferon-gamma/pharmacology , Killer Cells, Natural/immunology , beta 2-Microglobulin/physiologyABSTRACT
The expression of HLA-G by malignant cells has been proposed as a tumor escape mechanism from immunosurveillance. However, although the inhibitory effect of HLA-G on antitumoral immune effectors has been documented in vitro, it remains to be resolved in vivo. In this context, the development of an animal model is now a priority to establish the proof of concept that an HLA-G(+) tumor cell develops and tolerizes the host antitumor immune response in vivo. In the present study, we provide the first in vivo evidence of such a role by a xenotumor model in mice based on the interactions between human HLA-G and the murine paired immunoglobulin-like receptor-B (PIR-B). We demonstrate that human tumor cells expressing HLA-G grow in an immunocompetent host by affecting both innate and adaptive immunity. Expansion of blood myeloid-derived CD11b(+)Gr1(+)PIR-B(+) suppressor cells, loss of peripheral T cells, and cytokinic balance in favor of Th2 versus Th1/Th17 constitute the main mechanisms by which HLA-G promotes tumor expansion. These data demonstrate for the first time that HLA-G plays a crucial role in in vivo tumor evasion. Finally, blocking HLA-G function by a specific Ab inhibits the in vivo development of the tumor, offering a new innovative therapeutic strategy in cancer.
Subject(s)
Cell Proliferation , HLA Antigens/immunology , Histocompatibility Antigens Class I/immunology , Monitoring, Immunologic , Myeloid Cells/immunology , Neoplasms/immunology , Th2 Cells/immunology , Tumor Escape , Adaptive Immunity , Animals , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/metabolism , Cell Line, Tumor , Cytokines/metabolism , Female , HLA Antigens/chemistry , HLA Antigens/genetics , HLA Antigens/metabolism , HLA-G Antigens , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Immunity, Innate , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Mice , Mice, Inbred Strains , Myeloid Cells/metabolism , Neoplasm Transplantation , Neoplasms/metabolism , Neoplasms/pathology , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptors, Immunologic/metabolism , Th1-Th2 Balance , Th2 Cells/metabolismABSTRACT
The non-classical HLA class I molecule HLA-G was initially shown to play a major role in feto-maternal tolerance. Since this discovery, it has been established that HLA-G is a tolerogenic molecule which participates to the control of the immune response. In this review, we summarize the recent advances on (1) the multiple structures of HLA-G, which are closely associated with their role in the inhibition of NK cell cytotoxicity, (2) the factors that regulate the expression of HLA-G and its receptors, (3) the mechanism of action of HLA-G at the immunological synapse and through trogocytosis, and (4) the generation of suppressive cells through HLA-G. Moreover, we also review recent findings on the non-immunological functions of HLA-G in erythropoiesis and angiogenesis.
Subject(s)
HLA Antigens/immunology , HLA Antigens/metabolism , Hematopoiesis , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Animals , Gene Expression Regulation , HLA Antigens/chemistry , HLA Antigens/genetics , HLA-G Antigens , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Humans , Immunity , Immunological Synapses/immunologyABSTRACT
Vγ9Vδ2 T cells play a crucial role in the antitumoral immune response through cytokine production and cytotoxicity. Although the expression of the immunomodulatory molecule HLA-G has been found in diverse tumors, its impact on Vγ9Vδ2 T-cell functions remains unknown. Here we showed that soluble HLA-G inhibits Vγ9Vδ2 T-cell proliferation without inducing apoptosis. Moreover, soluble HLA-G inhibited the Vγ9Vδ2 T-cell production of IFN-γ induced by phosphoantigen stimulation. The reduction in Vγ9Vδ2 T-cell IFN-γ production was also induced by membrane-bound or soluble HLA-G expressed by tumor cell lines. Finally, primary tumor cells inhibited Vγ9Vδ2 T-cell proliferation and IFN-γ production through HLA-G. In this context, HLA-G impaired Vγ9Vδ2 T-cell cytotoxicity by interacting with ILT2 inhibitory receptor. These data demonstrate that HLA-G inhibits the anti-tumoral functions of Vγ9Vδ2 T cells and imply that treatments targeting HLA-G could optimize Vγ9Vδ2 T-cell-mediated immunotherapy of cancer.
Subject(s)
Cytotoxicity, Immunologic/immunology , HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Immunotherapy , Lymphocyte Activation/immunology , Melanoma/immunology , Receptors, Antigen, T-Cell, gamma-delta/antagonists & inhibitors , T-Lymphocytes/immunology , Antigens, CD/metabolism , Apoptosis , Blotting, Western , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , HLA-G Antigens , Humans , Interferon-gamma/metabolism , Leukocyte Immunoglobulin-like Receptor B1 , Melanoma/metabolism , Melanoma/therapy , Membrane Glycoproteins/metabolism , Receptors, Antigen, T-Cell, gamma-delta/metabolism , Receptors, Immunologic/metabolism , Receptors, KIR2DL4/metabolism , T-Lymphocytes/metabolism , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
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.
Subject(s)
Epidermis , Keratinocytes , ErbB Receptors , Humans , Inflammation , Integrin alpha6ABSTRACT
Tumor spheroids play an increasingly important role in cancer research. Their ability to recapitulate crucial features of tumor biology that are lost in the classically used 2D models along with their relative simplicity and handiness have made them the most studied 3D tumor model. Their application as a theranostic tool or as a means to study tumor-host interaction is now well-established in various cancers. However, their use in the field of Renal Cell Carcinoma (RCC) remains very limited. The aim of this work is to present methods to implement a basic RCC spheroid model. These methods cover the steps from RCC tumor dissociation to spheroid infiltration by immune cells. We present a protocol for RCC dissociation using Liberase TM and introduce a culture medium containing Epithelial Growth Factor and Hydrocortisone allowing for faster growth of RCC primary cells. We show that the liquid overlay technique allows for the formation of spheroids from cell lines and from primary cultures. We present a method using morphological criteria to select a homogeneous spheroid population based on a Fiji macro. We then show that spheroids can be infiltrated by PBMCs after activation with OKT3 or IL-15. Finally, we provide an example of application by implementing an immune spheroid killing assay allowing observing increased spheroid destruction after treatment with PD-1 inhibitors. Thus the straightforward methods presented here allow for efficient spheroid formation for a simple RCC 3D model that can be standardized and infused with immune cells to study immunotherapies.
ABSTRACT
Background: Placental malaria (PM) is associated with a higher susceptibility of infants to Plasmodium falciparum (Pf) malaria. A hypothesis of immune tolerance has been suggested but no clear explanation has been provided so far. Our goal was to investigate the involvement of inhibitory receptors LILRB1 and LILRB2, known to drive immune evasion upon ligation with pathogen and/or host ligands, in PM-induced immune tolerance. Method: Infants of women with or without PM were enrolled in Allada, southern Benin, and followed-up for 24 months. Antibodies with specificity for five blood stage parasite antigens were quantified by ELISA, and the frequency of immune cell subsets was quantified by flow cytometry. LILRB1 or LILRB2 expression was assessed on cells collected at 18 and 24 months of age. Findings: Infants born to women with PM had a higher risk of developing symptomatic malaria than those born to women without PM (IRR=1.53, p=0.040), and such infants displayed a lower frequency of non-classical monocytes (OR=0.74, p=0.01) that overexpressed LILRB2 (OR=1.36, p=0.002). Moreover, infants born to women with PM had lower levels of cytophilic IgG and higher levels of IL-10 during active infection. Interpretation: Modulation of IgG and IL-10 levels could impair monocyte functions (opsonisation/phagocytosis) in infants born to women with PM, possibly contributing to their higher susceptibility to malaria. The long-lasting effect of PM on infants' monocytes was notable, raising questions about the capacity of ligands such as Rifins or HLA-I molecules to bind to LILRB1 and LILRB2 and to modulate immune responses, and about the reprogramming of neonatal monocytes/macrophages.
Subject(s)
Antimalarials , Malaria, Falciparum , Membrane Glycoproteins , Placenta , Receptors, Immunologic , Antibodies, Protozoan , Female , Humans , Immunoglobulin G/blood , Infant , Infant, Newborn , Interleukin-10 , Leukocyte Immunoglobulin-like Receptor B1/genetics , Leukocyte Immunoglobulin-like Receptor B1/immunology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Monocytes/metabolism , Placenta/parasitology , Plasmodium falciparum , Pregnancy , Receptors, Immunologic/genetics , Receptors, Immunologic/immunologyABSTRACT
BACKGROUND: Survival after lung transplantation (LTx) still remains limited by chronic lung allograft dysfunction (CLAD), thought to represent a form of chronic rejection. We investigated whether the immune checkpoint HLA-G/ILT2 expressed by peripheral T-cell subpopulations could predict CLAD. METHODS: We used data for 150 LTx recipients from COLT (Cohort-For-Lung-Transplantation) cohort with ≥1 available blood sample at 1-, 6-, or 12-months post-Tx. Analysis of T cells by flow cytometry focused on the ILT2 receptor of HLA-G and other markers (CD57, CD25, CD127). T-cell subset analyses compared stable patients and those with CLAD at 3 years post-LTx. RESULTS: With data for 78 stable and 72 CLAD patients, among 21 T-cell subsets expressing ILT2, only CD4+CD57+ILT2+ T cells were associated with outcome. At 1-month post-Tx, low proportion of CD4+CD57+ILT2+ T cells was associated with reduced 3-year incidence of CLAD (CD4+CD57+ILT2+ T cells ≤ first IQR [25%] vs > first IQR, log-rank test, p = 0.028). Furthermore, the incidence of CLAD was higher with >2.6- vs ≤2.6-fold increased proportion of CD4+CD57+ILT2+ T cells over the first year post-LTx (3-year freedom frequencies: 27% [95%CI: 8-50] vs 64% [95%CI: 48-77] (log-rank test, p = 0.014). On multivariable analysis, increased proportion of CD4+CD57+ILT2+ T cells over the first year predicted CLAD (hazard ratio 1.25; 95%CI: 1.09-1.44; p = 0.001). Focusing on CD4+CD57+ILT2+ T cells, we demonstrated ex vivo that they are cytotoxic CD4+ T cells, selectively inhibited by HLA-G. CONCLUSIONS: Our data suggest that an early increase of CD4+CD57+ILT2+ T cells after LTx may be associated with CLAD onset.
Subject(s)
HLA-G Antigens , Lung Transplantation , Allografts , Humans , Lung , T-LymphocytesABSTRACT
CTLA4-Ig (Belatacept) is a new recombinant molecule that interferes with the signal of T lymphocyte activation and prevents acute rejection after renal transplantation. HLA-G acts as a naturally tolerogenic molecule in humans. In this study, we analyzed whether HLA-G contributes to CTLA4-Ig-mediated graft acceptance. Our results demonstrate that patients treated with CTLA4-Ig displayed significantly higher soluble HLA-G (sHLA-G) plasma concentrations (72 +/- 14 ng/ml) than patients treated with calcineurin inhibitors (5 +/- 1 ng/ml) or healthy donors (5 +/- 5 ng/ml). Notably, sHLA-G purified from plasma of CTLA4-Ig-treated patients was biologically active as it inhibited allogeneic T cell proliferation in vitro. Dendritic cells (DC) were identified as one of the cellular sources of sHLA-G in CTLA4-Ig-treated patients. Supporting this observation, we showed that DC generated in vitro in presence of CTLA4-Ig released sHLA-G in response to allostimulation. These CTLA4-Ig-treated DC acted as tolerogenic APC through sHLA-G secretion as they suppressed T cell alloproliferation, which could be restored by using a neutralizing anti-HLA-G Ab. These data define a novel pathway by which CTLA4-Ig immunomodulates allogenic response through posttranscriptional regulation of HLA-G expression in DC. CTLA4-Ig-mediated HLA-G release appears as a critical factor in T cell alloresponse inhibition, thereby contributing to the immunosuppressive effect and graft acceptance.
Subject(s)
Dendritic Cells/drug effects , Dendritic Cells/immunology , HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Immunoconjugates/pharmacology , Immunosuppressive Agents/pharmacology , Kidney Transplantation/immunology , Abatacept , Adult , Blotting, Western , Dendritic Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Graft Rejection/immunology , Graft Rejection/prevention & control , HLA Antigens/immunology , HLA-G Antigens , Histocompatibility Antigens Class I/immunology , Humans , Immunohistochemistry , Male , Middle Aged , RNA Processing, Post-Transcriptional , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Although the role of epidermal cells in skin regeneration has been extensively documented, their functions in immunity and tolerance mechanisms are largely underestimated. The aim of the present review was to outline the state of knowledge on resident immune cells of hematopoietic origin hosted in the epidermis, and then to focus on the involvement of keratinocytes in the complex skin immune networks acting in homeostasis and regeneration conditions. Based on this knowledge, the mechanisms of immune tolerance are reviewed. In particular, strategies based on immunosuppression mediated by HLA-G are highlighted, as recent advances in this field open up perspectives in epidermis-substitute bioengineering for temporary and permanent skin replacement strategies.
Subject(s)
HLA-G Antigens/immunology , Keratinocytes/immunology , Skin/immunology , Animals , Genetic Therapy , Homeostasis , Humans , Immune Tolerance , Skin/cytologyABSTRACT
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.