Immune microniches shape intestinal Treg function.

The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens1,2. Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in inflammatory disorders. Although cell and molecular interactions that control T regulatory (Treg) cell development and function have been identified3,4, less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive Treg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing5-7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and inflammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria-but not embedded lymphoid aggregates-is the key microniche that supports effector Treg (eTreg) cell function. eTreg cells are stable once their niche is established; however, unleashing inflammation breaks down compartmentalization, leading to dominance of CD103+SIRPα+ dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the lamina propria and identify receptor-ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies.

Cellular strategies to induce immune tolerance after liver transplantation: Clinical perspectives.

Liver transplantation (LT) has become the most efficient treatment for pediatric and adult end-stage liver disease and the survival time after transplantation is becoming longer due to the development of surgical techniques and perioperative management. However, long-term side-effects of immunosuppressants, like infection, metabolic disorders and malignant tumor are gaining more attention. Immune tolerance is the status in which LT recipients no longer need to take any immunosuppressants, but the liver function and intrahepatic histology maintain normal. The approaches to achieve immune tolerance after transplantation include spontaneous, operational and induced tolerance. The first two means require no specific intervention but withdrawing immunosuppressant gradually during follow-up. No clinical factors or biomarkers so far could accurately predict who are suitable for immunosuppressant withdraw after transplantation. With the understanding to the underlying mechanisms of immune tolerance, many strategies have been developed to induce tolerance in LT recipients. Cellular strategy is one of the most promising methods for immune tolerance induction, including chimerism induced by hematopoietic stem cells and adoptive transfer of regulatory immune cells. The safety and efficacy of various cell products have been evaluated by prospective preclinical and clinical trials, while obstacles still exist before translating into clinical practice. Here, we will summarize the latest perspectives and concerns on the clinical application of cellular strategies in LT recipients.

Regulatory T cell-mediated immunosuppression orchestrated by cancer: towards an immuno-genomic paradigm for precision medicine.

Accumulating evidence indicates that aberrant signalling stemming from genetic abnormalities in cancer cells has a fundamental role in their evasion of antitumour immunity. Immune escape mechanisms include enhanced expression of immunosuppressive molecules, such as immune-checkpoint proteins, and the accumulation of immunosuppressive cells, including regulatory T (Treg) cells, in the tumour microenvironment. Therefore, Treg cells are key targets for cancer immunotherapy. Given that therapies targeting molecules predominantly expressed by Treg cells, such as CD25 or GITR, have thus far had limited antitumour efficacy, elucidating how certain characteristics of cancer, particularly genetic abnormalities, influence Treg cells is necessary to develop novel immunotherapeutic strategies. Hence, Treg cell-targeted strategies based on the particular characteristics of cancer in each patient, such as the combination of immune-checkpoint inhibitors with molecularly targeted agents that disrupt the immunosuppressive networks mediating Treg cell recruitment and/or activation, could become a new paradigm of cancer therapy. In this Review, we discuss new insights on the mechanisms by which cancers generate immunosuppressive networks that attenuate antitumour immunity and how these networks confer resistance to cancer immunotherapy, with a focus on Treg cells. These insights lead us to propose the concept of 'immuno-genomic precision medicine' based on specific characteristics of cancer, especially genetic profiles, that correlate with particular mechanisms of tumour immune escape and might, therefore, inform the optimal choice of immunotherapy for individual patients.

Single-cell profiling reveals immune disturbances landscape and HLA-F-mediated immune tolerance at the maternal-fetal interface in preeclampsia.

Background: Preeclampsia is a pregnancy-specific disorder that always causes maternal and fetal serious adverse outcome. Disturbances in maternal immune tolerance to embryo at the maternal-fetal interface (MFI) may be associated with preeclampsia onset. Recent studies have revealed the reduced expression pattern of HLA-F at the MFI in preeclampsia, while the mechanism of it mediating maternal fetal immune tolerance has not been revealed. Methods: Single-cell RNA sequencing on placental decidua was performed to reveal the immune disturbances landscape at the MFI in preeclampsia. Human Jar cells and NK-92MI cells were employed to study the role of HLA-F in trophoblasts and lymphocyte. Results: A total of 101,250 cells were classified into 22 cell clusters. Disease-related IGFBP1+SPP1+ extracellular villus trophoblast (EVT) was identified in the preeclamptic placental decidua, accompanied by newly discovered immune cellular dysfunction such as reduced ribosomal functions of NK populations and abnormal expression of antigen-presenting molecules in most cell clusters. Certain genes that are characteristic of the intermediate stage of myeloid or EVT cell differentiation were found to have unexplored but important functions in the pathogenesis of preeclampsia; specifically, we detected enhanced cell cross-talk between IGFBP1+SPP1+ EVT2 or SPP1+M1 cells and their receptor cell populations at the MFI of PE patients compared to controls. With respect to HLA-F, mIF staining confirmed its reduced expression in PE samples compared to controls. Over-expression of HLA-F in Jar cells promoted cell proliferation, invasion, and migration while under-expression had the opposite effect. In NK-92MI cells, over-expression of HLA-F increased the secretion of immunoregulation cytokines such as CSF1 and CCL22, and promoted adaptive NKG2C+NK cell transformation. Conclusions: We revealed the immune disturbance landscape at the MFI in preeclampsia. Our findings regarding cellular heterogeneity and immune cellular dysfunction, as revealed by scRNA-seq, and the function of HLA-F in cells provide new perspectives for further investigation of their roles in the pathogenesis of preeclampsia, and then provide potential new therapeutic target.

[The reasons for the formation of tolerance to food antigens].

One of the main issues of the peculiarities of the immune reactions of the gastrointestinal tract is the mechanisms of ensuring tolerance to food antigens. Concentrations of antibodies to food antigens actually reflect the state of the intestinal mucosa barrier function, and the degree of penetration of antigens into the blood determines the level of immune response to them. The aim of the study was to determine the risk criteria for violation of tolerance to food antigens. Material and methods. The study included the results of a survey and examination of 1334 adults living in the north of the European part of the Russian Federation, including 1100 born in the North, of which 970 were women and 364 were men. The average age of the respondents was 45.5±1.0 years. The comparison group consisted of 344 patients with pathology of the gastrointestinal tract who applied to the medical company "Biocor". The content of immunoglobulins (Ig) G to food antigens, total IgA, cytokines (tumor necrosis factor α, interleukin-6, interleukin-4) in blood serum were determined by enzyme immunoassay. Results. Rural residents often (more than 28%) have elevated concentrations of IgG to potato, river fish, wheat and rye antigens. Urban residents have the most pronounced decrease in tolerance to food antigens of chicken, cod, beef and pork. In healthy individuals, elevated (>100 ME/ml) concentrations of antibodies to meat products are recorded in the range of 11.3-13.9%, to dairy antigens - 11.5-14.1%, cereals - 11.9-13.4%. Slightly less frequently, elevated concentrations of antibodies to fish antigens (7.5-10.1%), vegetables (3.8-7.0%) and fruits (4.9-6.5%) are detected. In inflammatory and oncological diseases of the gastrointestinal tract, the content of antibodies to food antigens increases sharply. On average, the frequency of impaired tolerance to food antigens in patients is 2.7-6.1 times higher than in healthy individuals. Conclusion. Violation of tolerance to food antigens is associated with an increase in blood pro-inflammatory cytokines, mainly interleukin-6. In practically healthy individuals, a decrease in tolerance to food antigens is associated with a deficiency of blood IgA. The risk criteria for violation of the diet or consumption of low-quality foods may be an increase in the frequency of detection of elevated concentrations of antibodies to meat products in 14.6±3.0%, fish - 10.7±2.3%, cereals - 13.7±1.6%, dairy products - 14.8±1.5%, vegetables - 7.8±2.4% and fruits - 6.9±5.8%.

Decoding foreign antigen tolerance.

Cell atlases of human tolerogenic milieus guide transformative immunotherapies.

Tissue CD14+CD8+ T cells reprogrammed by myeloid cells and modulated by LPS.

The liver is bathed in bacterial products, including lipopolysaccharide transported from the intestinal portal vasculature, but maintains a state of tolerance that is exploited by persistent pathogens and tumours1-4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+ T cells compartmentalized in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins and are enriched in close proximity to CD14high myeloid cells in hepatic zone 2. CD14+CD8+ T cells preferentially accumulate within the donor pool in liver allografts, among hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+ T cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10 and IL-2 production ex vivo, and enhanced antiviral/anti-tumour effector function after TCR engagement. This CD14+CD8+ T cell profile can be recapitulated by the acquisition of membrane proteins-including the lipopolysaccharide receptor complex-from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T cells in vitro. CD14+CD8+ T cells express integrins and chemokine receptors that favour interactions with the local stroma, which can promote their induction through CXCL12. Lipopolysaccharide can also increase the frequency of CD14+CD8+ T cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+ T cells with immunomodulatory ability.

Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy.

PURPOSE: Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. EXPERIMENTAL DESIGN: Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. RESULTS: It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. CONCLUSION: The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.

Tryptophan degradation enzymes and Angiotensin (1-7) expression in human placenta.

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are key enzymes for tryptophan degradation, regulating immune tolerance during pregnancy. The intrauterine renin-angiotensin system is also involved in the progression of a healthy pregnancy. Angiotensin(1-7) maintains the integrity of fetal membranes via counteracting the pro-inflammatory actions of Angiotensin II. No data are available on placental Angiotensin(1-7) co-expression with TDO. We aimed to characterize TDO mRNA expression and its localization in different areas of the placenta of physiological pregnancies delivered at term; its co-expression with Angiotensin(1-7) and its correlation with the plasma kynurenine/tryptophan (Kyn/Trp) ratio was investigated. This prospective observational study included a nonconsecutive series of 20 singleton uncomplicated pregnancies delivered vaginally. TDO mRNA was expressed in both maternal and fetal sides of the placentas and TDO protein also in the villi and it was co-expressed with IDO1 in almost half of the placental cells at these sites. The percentage of TDO+ and IDO1+ cells appeared to be influenced by maternal pre-gestational smoking and newborn weight. A strong correlation was found between the percentage of TDO+ and IDO1+ cells in the villi. TDO+ cells also expressed Angiotensin(1-7), with a higher percentage on the fetal side and in the villi compared to the maternal one. Kyn/Trp plasma ratio was not correlated with IDO and TDO expression nor with the patient's characteristics. Collectively, our data indicate that TDO is detectable in placental tissue and is co-expressed with IDO and with Angiotensin(1-7)+ on the fetal side and in the villi.

Immune tolerance of food is mediated by layers of CD4+ T cell dysfunction.

Gastrointestinal health depends on the adaptive immune system tolerating the foreign proteins in food1,2. This tolerance is paradoxical because the immune system normally attacks foreign substances by generating inflammation. Here we addressed this conundrum by using a sensitive cell enrichment method to show that polyclonal CD4+ T cells responded to food peptides, including a natural one from gliadin, by proliferating weakly in secondary lymphoid organs of the gut-liver axis owing to the action of regulatory T cells. A few food-specific T cells then differentiated into T follicular helper cells that promoted a weak antibody response. Most cells in the expanded population, however, lacked canonical T helper lineage markers and fell into five subsets dominated by naive-like or T follicular helper-like anergic cells with limited capacity to form inflammatory T helper 1 cells. Eventually, many of the T helper lineage-negative cells became regulatory T cells themselves through an interleukin-2-dependent mechanism. Our results indicate that exposure to food antigens causes cognate CD4+ naive T cells to form a complex set of noncanonical hyporesponsive T helper cell subsets that lack the inflammatory functions needed to cause gut pathology and yet have the potential to produce regulatory T cells that may suppress it.

Glycans as a key factor in self and nonself discrimination: impact on the breach of immune tolerance.

Glycans are carbohydrates that are made by all organisms and covalently conjugated to other biomolecules. Glycans cover the surface of both human cells and pathogens and are fundamental to defining the identity of a cell or an organism, thereby contributing to discriminating self from nonself. As such, glycans are a class of 'Self-Associated Molecular Patterns' that can fine-tune host inflammatory processes. In fact, glycans can be sensed and recognized by a variety of glycan-binding proteins (GBP) expressed by immune cells, such as galectins, siglecs, and C-type lectins, which recognize changes in the cellular glycosylation, instructing both pro-inflammatory and anti-inflammatory responses. In this review, we introduce glycans as cell-identification structures, discussing how glycans modulate host-pathogen interactions and how they can fine-tune inflammatory processes associated with infection, inflammation and autoimmunity. Finally, from the clinical standpoint, we discuss how glycoscience research can benefit life sciences and clinical medicine by providing a source of valuable biomarkers and therapeutic targets for immunity.

Tryptophan-derived microbial metabolites activate the aryl hydrocarbon receptor in tumor-associated macrophages to suppress anti-tumor immunity.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

Autoreactive antibodies control blood glucose by regulating insulin homeostasis.

Homeostasis of metabolism by hormone production is crucial for maintaining physiological integrity, as disbalance can cause severe metabolic disorders such as diabetes mellitus. Here, we show that antibody-deficient mice and immunodeficiency patients have subphysiological blood glucose concentrations. Restoring blood glucose physiology required total IgG injections and insulin-specific IgG antibodies detected in total IgG preparations and in the serum of healthy individuals. In addition to the insulin-neutralizing anti-insulin IgG, we identified two fractions of anti-insulin IgM in the serum of healthy individuals. These autoreactive IgM fractions differ in their affinity to insulin. Interestingly, the low-affinity IgM fraction (anti-insulin IgMlow) neutralizes insulin and leads to increased blood glucose, whereas the high-affinity IgM fraction (anti-insulin IgMhigh) protects insulin from neutralization by anti-insulin IgG, thereby preventing blood glucose dysregulation. To demonstrate that anti-insulin IgMhigh acts as a protector of insulin and counteracts insulin neutralization by anti-insulin IgG, we expressed the variable regions of a high-affinity anti-insulin antibody as IgG and IgM. Remarkably, the recombinant anti-insulin IgMhigh normalized insulin function and prevented IgG-mediated insulin neutralization. These results suggest that autoreactive antibodies recognizing insulin are key regulators of blood glucose and metabolism, as they control the concentration of insulin in the blood. Moreover, our data suggest that preventing autoimmune damage and maintaining physiological homeostasis requires adaptive tolerance mechanisms generating high-affinity autoreactive IgM antibodies during memory responses.

Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Lin28B-high breast cancer cells promote immune suppression in the lung pre-metastatic niche via exosomes and support cancer progression.

The formation of pre-metastatic niche is a key step in the metastatic burden. The pluripotent factor Lin28B is frequently expressed in breast tumors and is particularly upregulated in the triple negative breast cancer subtype. Here, we demonstrate that Lin28B promotes lung metastasis of breast cancer by building an immune-suppressive pre-metastatic niche. Lin28B enables neutrophil recruitment and N2 conversion. The N2 neutrophils are then essential for immune suppression in pre-metastatic lung by PD-L2 up-regulation and a dysregulated cytokine milieu. We also identify that breast cancer-released exosomes with low let-7s are a prerequisite for Lin28B-induced immune suppression. Moreover, Lin28B-induced breast cancer stem cells are the main sources of low-let-7s exosomes. Clinical data further verify that high Lin28B and low let-7s in tumors are both indicators for poor prognosis and lung metastasis in breast cancer patients. Together, these data reveal a mechanism by which Lin28B directs the formation of an immune-suppressive pre-metastatic niche.

Mesenchymal stem cells transfer mitochondria to allogeneic Tregs in an HLA-dependent manner improving their immunosuppressive activity.

Cell-based immunotherapies can provide safe and effective treatments for various disorders including autoimmunity, cancer, and excessive proinflammatory events in sepsis or viral infections. However, to achieve this goal there is a need for deeper understanding of mechanisms of the intercellular interactions. Regulatory T cells (Tregs) are a lymphocyte subset that maintain peripheral tolerance, whilst mesenchymal stem cells (MSCs) are multipotent nonhematopoietic progenitor cells. Despite coming from different origins, Tregs and MSCs share immunoregulatory properties that have been tested in clinical trials. Here we demonstrate how direct and indirect contact with allogenic MSCs improves Tregs' potential for accumulation of immunosuppressive adenosine and suppression of conventional T cell proliferation, making them more potent therapeutic tools. Our results also demonstrate that direct communication between Tregs and MSCs is based on transfer of active mitochondria and fragments of plasma membrane from MSCs to Tregs, an event that is HLA-dependent and associates with HLA-C and HLA-DRB1 eplet mismatch load between Treg and MSC donors.

Long Noncoding RNA HOTAIRM1 Promotes Immunosuppression in Sepsis by Inducing T Cell Exhaustion.

Sepsis is an acute life-threatening disorder associated with multiorgan dysfunction that remains the leading cause of death in intensive care units. As sepsis progresses, it causes prolonged immunosuppression, which results in sustained mortality, morbidity, and susceptibility to secondary infections. Using a mouse model of sepsis, we found that the long noncoding RNA HOTAIRM1 (HOXA transcript antisense RNA myeloid-specific 1) was highly expressed in mice during the late phase of sepsis. The upregulation of HOTAIRM1 was induced by Notch/Hes1 activation and, moreover, was critical for the formation of an immunosuppressive microenvironment. HOTAIRM1 induced T cell exhaustion by increasing the percentage of PD-1+ T cells and regulatory T cells, accompanied by elevated PD-L1. Blockade of either Notch/Hes1 signaling or HOTAIRM1 inhibited T cell exhaustion in late sepsis, having alleviated lung injury and improved survival of mice. Further mechanistic studies identified HOXA1 as a key transcription factor targeted by HOTAIRM1 to regulate PD-L1 expression in lung alveolar epithelial cells. These results implicated that the Notch/Hes1/HOTAIRM1/HOXA1/PD-L1 axis was critical for sepsis-induced immunosuppression and could be a potential target for sepsis therapies.