Overcoming resistance to immune checkpoint blockade in liver cancer - stronger together?

Primary liver cancer, represented mainly by hepatocellular carcinoma (HCC) and intrahepatic cholangiocellular carcinoma (CCA), is one of the most common and deadliest tumors worldwide. While surgical resection or liver transplantation are the best option in early disease stages, these tumors often present in advanced stages and systemic treatment is required to improve survival time. The emergence of immune checkpoint inhibitor therapy has had a positive impact especially on the treatment of advanced cancers, thereby establishing immunotherapy as part of first-line treatment in HCC and CCA. Nevertheless, low response rates reflect on the usually cold or immunosuppressed tumor microenvironment of primary liver cancer. In this review, we aim to summarize mechanisms of resistance leading to tumor immune escape with a special focus on the composition of tumor microenvironment in both HCC and CCA, also reflecting on recent important developments in ICI combination therapy. Furthermore, we discuss how combination of immune checkpoint inhibitors with established primary liver cancer treatments (e.g. multikinase inhibitors and chemotherapy) as well as more complex combinations with state-of-the-art therapeutic concepts may re-shape the tumor microenvironment, leading to higher response rates and long-lasting anti-tumor immunity for primary liver cancer patients.

CCL18 aggravates atherosclerosis by inducing CCR6-dependent T-cell influx and polarization.

Introduction: The CC chemokine ligand 18 (CCL18) is a chemokine highly expressed in chronic inflammation in humans. Recent observations of elevated CCL18 plasma levels in patients with acute cardiovascular syndromes prompted an investigation into the role of CCL18 in the pathogenesis of human and mouse atherosclerosis. Methods and results: CCL18 was profoundly upregulated in ruptured human atherosclerotic plaque, particularly within macrophages. Repeated administration of CCL18 in Western-type diet-fed ApoE -/- mice or PCSK9mut-overexpressing wild type (WT) mice led to increased plaque burden, enriched in CD3+ T cells. In subsequent experimental and molecular modeling studies, we identified CCR6 as a functional receptor mediating CCL18 chemotaxis, intracellular Ca2+ flux, and downstream signaling in human Jurkat and mouse T cells. CCL18 failed to induce these effects in vitro in murine spleen T cells with CCR6 deficiency. The ability of CCR6 to act as CCL18 receptor was confirmed in vivo in an inflammation model, where subcutaneous CCL18 injection induced profound focal skin inflammation in WT but not in CCR6-/- mice. This inflammation featured edema and marked infiltration of various leukocyte subsets, including T cells with a Th17 signature, supporting CCR6's role as a Th17 chemotactic receptor. Notably, focal overexpression of CCL18 in plaques was associated with an increased presence of CCR6+ (T) cells. Discussion: Our studies are the first to identify the CCL18/CCR6 axis as a regulator of immune responses in advanced murine and human atherosclerosis.

Fluorochrome-dependent specific changes in spectral profiles using different compensation beads or primary cells in full spectrum cytometry.

Full spectrum flow cytometry is a powerful tool for immune monitoring on a single-cell level and with currently available machines, panels of 40 or more markers per sample are possible. However, with an increased panel size, spectral unmixing issues arise, and appropriate single stain reference controls are required for accurate experimental results and to avoid unmixing errors. In contrast to conventional flow cytometry, full spectrum flow cytometry takes into account even minor differences in spectral signatures and requires the full spectrum of each fluorochrome to be identical in the reference control and the fully stained sample to ensure accurate and reliable results. In general, using the cells of interest is considered optimal, but certain markers may not be expressed at sufficient levels to generate a reliable positive control. In this case, compensation beads show some significant advantages as they bind a consistent amount of antibody independent of its specificity. In this study, we evaluated two types of manufactured compensation beads for use as reference controls for 30 of the most commonly used and commercially available fluorochromes in full spectrum cytometry and compared them to human and murine primary leukocytes. While most fluorochromes show the same spectral profile on beads and cells, we demonstrate that specific fluorochromes show a significantly different spectral profile depending on which type of compensation beads is used, and some fluorochromes should be used on cells exclusively. Here, we provide a list of important considerations when selecting optimal reference controls for full spectrum flow cytometry.

Hepatic inflammatory responses in liver fibrosis.

Chronic liver diseases such as nonalcoholic fatty liver disease (NAFLD) or viral hepatitis are characterized by persistent inflammation and subsequent liver fibrosis. Liver fibrosis critically determines long-term morbidity (for example, cirrhosis or liver cancer) and mortality in NAFLD and nonalcoholic steatohepatitis (NASH). Inflammation represents the concerted response of various hepatic cell types to hepatocellular death and inflammatory signals, which are related to intrahepatic injury pathways or extrahepatic mediators from the gut-liver axis and the circulation. Single-cell technologies have revealed the heterogeneity of immune cell activation concerning disease states and the spatial organization within the liver, including resident and recruited macrophages, neutrophils as mediators of tissue repair, auto-aggressive features of T cells as well as various innate lymphoid cell and unconventional T cell populations. Inflammatory responses drive the activation of hepatic stellate cells (HSCs), and HSC subsets, in turn, modulate immune mechanisms via chemokines and cytokines or transdifferentiate into matrix-producing myofibroblasts. Current advances in understanding the pathogenesis of inflammation and fibrosis in the liver, mainly focused on NAFLD or NASH owing to the high unmet medical need, have led to the identification of several therapeutic targets. In this Review, we summarize the inflammatory mediators and cells in the diseased liver, fibrogenic pathways and their therapeutic implications.

Intratumoral dendritic cells and T cells predict survival in gastroenteropancreatic neuroendocrine neoplasms.

Clinical management of gastroenteropancreatic neuroendocrine neoplasms remains challenging. We recently introduced the FMS-like tyrosine kinase 3 ligand (FLT3LG) as a possible biomarker for a proinflammatory tumor microenvironment. Here, we put a spotlight on the quantitative assessment of classical dendritic cells (cDC) and T cells in the context of FLT3LG mRNA levels in a retrospective study on neuroendocrine tumor (NET) G2/G3 and neuroendocrine carcinoma (NEC) of pancreatic and gastric origin. The abundance of cDC and T cells and their relevant subpopulations were determined by immunofluorescent staining and correlated with FLT3LG mRNA levels as well as clinical outcomes. Immune cell counts attested to highly variable infiltration densities. Samples with the presence of cDC or high numbers of T cells exhibited increased FLT3LG expression. Abundance of cDC, defined as HLA-DR+CD11c+ cells with CLEC9a (cDC1) or CD1c (cDC2), as well as T cells correlated with FLT3LG mRNA levels and predicted disease-specific survival. Combining FLT3LG and T cell counts further improved this prediction. Therefore, tumor-infiltrating cDC and T cells are prognostic markers in NET G2/G3 or NEC and FLT3LG mRNA may serve as a simple-to-use biomarker for a quantitative estimate of their abundance, mandating prospective evaluation in the context of immune-targeted therapies.

Resolving 31 colors on a standard 3-laser full spectrum flow cytometer for immune monitoring of human blood samples.

Immune monitoring of patients on a single-cell level is becoming increasingly important in various diseases. Due to the often very limited availability of human specimens and our increased understanding of the immune systems there is an increasing demand to analyze as many markers as possible simultaneously in one panel. Full spectrum flow cytometry is emerging as a powerful tool for immune monitoring since 5-laser instruments enable characterization of 40 parameters or more in a single sample. Nevertheless, even if only machines with fewer lasers are available, development of novel fluorophore families enables increasing panel sizes. Here, we demonstrate that careful panel design enables the use of 31-color panels on a 3-laser Cytek® Aurora cytometer for analyzing human peripheral blood leukocytes, without the need for custom configuration and using only commercially available fluorochromes. The panel presented here should serve as an example of a 31-fluorochrome combination that can be resolved on a 3-laser full spectrum cytometer and that can be adapted to comprise other (and possibly more) markers of interest depending on the research focus.

Hepatic Stellate Cells: Dictating Outcome in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a fast growing, chronic liver disease affecting ∼25% of the global population. Nonalcoholic fatty liver disease severity ranges from the less severe simple hepatic steatosis to the more advanced nonalcoholic steatohepatitis (NASH). The presence of NASH predisposes individuals to liver fibrosis, which can further progress to cirrhosis and hepatocellular carcinoma. This makes hepatic fibrosis an important indicator of clinical outcomes in patients with NASH. Hepatic stellate cell activation dictates fibrosis development during NASH. Here, we discuss recent advances in the analysis of the profibrogenic pathways and mediators of hepatic stellate cell activation and inactivation, which ultimately determine the course of disease in nonalcoholic fatty liver disease/NASH.

Elevated Serum Levels of CCL23 Are Associated with Poor Outcome after Resection of Biliary Tract Cancer.

Background: Surgical tumor resection is the only potentially curative treatment option for patients with biliary tract cancer (BTC). However, 5-year survival rates are still below 50% mainly due to tumor recurrence. The preoperative identification of ideal surgical candidates has remained a major challenge and easily accessible algorithms including parameters of the individual tumor biology are missing. Chemokine (C-C motif) ligand 23 (CCl23) has been associated with tumor progression in hepatocellular carcinoma (HCC), but its role in the context of BTC is largely unknown. Here, we evaluated circulating levels of CCL23 as potential diagnostic and prognostic biomarker in patients with resectable BTC. Methods: CCl23 serum levels were analyzed by multiplex immunoassay in a cohort of 119 BTC patients receiving surgical tumor resection as well as 50 healthy control samples and 11 patients with primary sclerosing cholangitis (PSC). Results: Baseline serum CCL23 levels were significantly elevated in BTC patients compared to PSC patients as well as healthy controls. CCL23 increased the diagnostic sensitivity and specificity of established tumor markers including CA19-9 and correlated with patients' age and makers of systemic inflammation. Elevated preoperative CCL23 levels were associated with a significantly impaired postoperative outcome. BTC patients with a preoperative CCL23 level above the optimal prognostic cut-off value of 702.4 pg/ml showed a median OS of only 110 days compared to 501 days for patients with low initial CCL23 levels. The prognostic value of circulating CCL23 was confirmed in Cox-regression analysis. Conclusion: Serum levels of CCL23 are elevated in patients with BTC, and high preoperative CCL23 levels were associated with an impaired postoperative survival. CCL23 serum levels could help to identify the ideal surgical candidates for BTC resection in the future.

Expanding cross-presenting dendritic cells enhances oncolytic virotherapy and is critical for long-term anti-tumor immunity.

Immunotherapies directly enhancing anti-tumor CD8+ T cell responses have yielded measurable but limited success, highlighting the need for alternatives. Anti-tumor T cell responses critically depend on antigen presenting dendritic cells (DC), and enhancing mobilization, antigen loading and activation of these cells represent an attractive possibility to potentiate T cell based therapies. Here we show that expansion of DCs by Flt3L administration impacts in situ vaccination with oncolytic Newcastle Disease Virus (NDV). Mechanistically, NDV activates DCs and sensitizes them to dying tumor cells through upregulation of dead-cell receptors and synergizes with Flt3L to promote anti-tumor CD8+ T cell cross-priming. In vivo, Flt3L-NDV in situ vaccination induces parallel amplification of virus- and tumor-specific T cells, including CD8+ T cells reactive to newly-described neoepitopes, promoting long-term tumor control. Cross-presenting conventional Type 1 DCs are indispensable for the anti-tumor, but not anti-viral, T cell response, and type I IFN-dependent CD4+ Th1 effector cells contribute to optimal anti-tumor immunity. These data demonstrate that mobilizing DCs to increase tumor antigen cross-presentation improves oncolytic virotherapy and that neoepitope-specific T cells can be induced without individualized, ex vivo manufactured vaccines.

Inflammatory Cytokines Associated with Diagnosis, Tumor Grade and Prognosis in Patients with Neuroendocrine Tumors.

Background and aims: Inflammatory cytokines represent diagnostic and prognostic biomarkers in manifold cancers. Recent data suggest a pivotal role of these cytokines in different biological processes involved in the development of neuroendocrine tumors (NETs). However, their role as biomarkers in NETs is only poorly understood. Methods: We analyzed serum concentrations of 13 inflammation-related cytokines at different time points in 43 patients with well-differentiated gastroenteropancreatic NETs (G1/G2) treated at Charité Berlin and compared them to 40 healthy controls. The results were correlated with clinical records. Results: Serum concentrations (Median (Interquartile Range (IQR)) in pg/mL) of IL-1ß (124 (82) vs. 68 (61) pg/mL; p = 0.0003), IL-6 (111(122) vs. 88 (32) pg/mL; p = 0.0086), IL-8 (1058 (768) vs. 210 (90) pg/mL; p < 0.0001), IL-18 (2936 (1723) vs. 1590 (704) pg/mL; p < 0.0001), and TNF (271 (260) vs. 42 (25) pg/mL; p < 0.0001) were significantly elevated in NET patients, whereas IL-10 (43 (44) vs. 105 (48) pg/mL; p < 0.0001) showed lower concentrations in NETs when compared to controls. Cytokine levels significantly correlated with tumor grade (IL-6; p = 0.0070), prevalence of distant metastasis (IL-18; p = 0.0313), and disease progression over time (IL-10; p = 0.0033) but not tumor location. Chromogranin A (CgA) and the NETest are currently used to monitor treatment response. A more accurate prediction could possibly be achieved by employing a subset of cytokines. Our data clearly warrants further functional investigation into the role of the immune response and cytokine release in NETs. Conclusion: A biologically plausible panel of cytokines might be added to the diagnostic and prognostic tools currently employed in patients with NETs. Combining different markers into a score would elevate diagnostic accuracy compared to single markers.

Combination of G-CSF and a TLR4 inhibitor reduce inflammation and promote regeneration in a mouse model of ACLF.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is characterised by high short-term mortality, systemic inflammation, and failure of hepatic regeneration. Its treatment is a major unmet medical need. This study was conducted to explore whether combining TAK-242, a Toll-like receptor-4 (TLR4) antagonist, with granulocyte-colony stimulating factor (G-CSF), could reduce inflammation whilst enhancing liver regeneration. METHODS: Two mouse models of ACLF were investigated. Chronic liver injury was induced by carbon tetrachloride; lipopolysaccharide (LPS) or galactosamine (GalN) were then administered as extrahepatic or hepatic insults, respectively. G-CSF and/or TAK-242 were administered daily. Treatment durations were 24 hours and 5 days in the LPS model and 48 hours in the GalN model. RESULTS: In a mouse model of LPS-induced ACLF, treatment with G-CSF was associated with significant mortality (66% after 48 hours vs. 0% without G-CSF). Addition of TAK-242 to G-CSF abrogated mortality (0%) and significantly reduced liver cell death, macrophage infiltration and inflammation. In the GalN model, both G-CSF and TAK-242, when used individually, reduced liver injury but their combination was significantly more effective. G-CSF treatment, with or without TAK-242, was associated with activation of the pro-regenerative and anti-apoptotic STAT3 pathway. LPS-driven ACLF was characterised by p21 overexpression, which is indicative of hepatic senescence and inhibition of hepatocyte regeneration. While TAK-242 treatment mitigated the effect on senescence, G-CSF, when co-administered with TAK-242, resulted in a significant increase in markers of hepatocyte regeneration. CONCLUSION: The combination of TAK-242 and G-CSF inhibits inflammation, promotes hepatic regeneration and prevents mortality in models of ACLF; thus, this combination could be a potential treatment option for ACLF. LAY SUMMARY: Acute-on-chronic liver failure is associated with severe liver inflammation and poor short-term survival. Therefore, effective treatments are urgently needed. Herein, we have shown, using mouse models, that the combination of granulocyte-colony stimulating factor (which can promote liver regeneration) and TAK-242 (which inhibits a receptor that plays a key role in inflammation) could be effective for the treatment of acute-on-chronic liver failure.

Elevated Flt3L Predicts Long-Term Survival in Patients with High-Grade Gastroenteropancreatic Neuroendocrine Neoplasms.

BACKGROUND: The clinical management of high-grade gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) is challenging due to disease heterogeneity, illustrating the need for reliable biomarkers facilitating patient stratification and guiding treatment decisions. FMS-like tyrosine kinase 3 ligand (Flt3L) is emerging as a prognostic or predictive surrogate marker of host tumoral immune response and might enable the stratification of patients with otherwise comparable tumor features. METHODS: We evaluated Flt3L gene expression in tumor tissue as well as circulating Flt3L levels as potential biomarkers in a cohort of 54 patients with GEP-NEN. RESULTS: We detected a prominent induction of Flt3L gene expression in individual G2 and G3 NEN, but not in G1 neuroendocrine tumors (NET). Flt3L mRNA expression levels in tumor tissue predicted the disease-related survival of patients with highly proliferative G2 and G3 NEN more accurately than the conventional criteria of grading or NEC/NET differentiation. High level Flt3L mRNA expression was associated with the increased expression of genes related to immunogenic cell death, lymphocyte effector function and dendritic cell maturation, suggesting a less tolerogenic (more proinflammatory) phenotype of tumors with Flt3L induction. Importantly, circulating levels of Flt3L were also elevated in high grade NEN and correlated with patients' progression-free and disease-related survival, thereby reflecting the results observed in tumor tissue. CONCLUSIONS: We propose Flt3L as a prognostic biomarker for high grade GEP-NEN, harnessing its potential as a marker of an inflammatory tumor microenvironment. Flt3L measurements in serum, which can be easily be incorporated into clinical routine, should be further evaluated to guide patient stratification and treatment decisions.

The Role of miRNA in the Pathophysiology of Neuroendocrine Tumors.

Neuroendocrine tumors (NETs) represent a tumor group that is both rare and heterogeneous. Prognosis is largely determined by the tumor grading and the site of the primary tumor and metastases. Despite intensive research efforts, only modest advances in diagnostic and therapeutic approaches have been achieved in recent years. For patients with non-respectable tumor stages, prognosis is poor. In this context, the development of novel diagnostic tools for early detection of NETs and prediction of tumor response to therapy as well as estimation of the overall prognosis would greatly improve the clinical management of NETs. However, identification of novel diagnostic molecules is hampered by an inadequate understanding of the pathophysiology of neuroendocrine malignancies. It has recently been demonstrated that microRNA (miRNA), a family of small RNA molecules with an established role in the pathophysiology of quite different cancer entities, may also play a role as a biomarker. Here, we summarize the available knowledge on the role of miRNAs in the development of NET and highlight their potential use as serum-based biomarkers in the context of this disease. We discuss important challenges currently preventing their use in clinical routine and give an outlook on future directions of miRNA research in NET.

In Situ Vaccination as a Strategy to Modulate the Immune Microenvironment of Hepatocellular Carcinoma.

Hepatocellular Carcinoma (HCC) is a highly prevalent malignancy that develops in patients with chronic liver diseases and dysregulated systemic and hepatic immunity. The tumor microenvironment (TME) contains tumor-associated macrophages (TAM), cancer-associated fibroblasts (CAF), regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) and is central to mediating immune evasion and resistance to therapy. The interplay between these cells types often leads to insufficient antigen presentation, preventing effective anti-tumor immune responses. In situ vaccines harness the tumor as the source of antigens and implement sequential immunomodulation to generate systemic and lasting antitumor immunity. Thus, in situ vaccines hold the promise to induce a switch from an immunosuppressive environment where HCC cells evade antigen presentation and suppress T cell responses towards an immunostimulatory environment enriched for activated cytotoxic cells. Pivotal steps of in situ vaccination include the induction of immunogenic cell death of tumor cells, a recruitment of antigen-presenting cells with a focus on dendritic cells, their loading and maturation and a subsequent cross-priming of CD8+ T cells to ensure cytotoxic activity against tumor cells. Several in situ vaccine approaches have been suggested, with vaccine regimens including oncolytic viruses, Flt3L, GM-CSF and TLR agonists. Moreover, combinations with checkpoint inhibitors have been suggested in HCC and other tumor entities. This review will give an overview of various in situ vaccine strategies for HCC, highlighting the potentials and pitfalls of in situ vaccines to treat liver cancer.

Lessons From Immune Checkpoint Inhibitor Trials in Hepatocellular Carcinoma.

The implementation of immune checkpoint inhibitors (ICI) into the clinical management of different malignancies has largely changed our understanding of cancer treatment. After having proven efficacy in different tumor entities such as malignant melanoma and lung cancer, ICI were intensively tested in the setting of hepatocellular carcinoma (HCC). Here they could achieve higher and more durable response rates compared to tyrosine-kinase inhibitors (TKI), that were sole standard of care for the last decade. Most recently, ICI treatment was approved in a first line setting of HCC, for cases not suitable for curative strategies. However, only a subset of patients benefits from ICI therapy, while others experience rapid tumor progression, worsening of liver function and poor prognosis. Efforts are being made to find immune characteristics that predict tumor responsiveness to ICI, but no reliable biomarker could be identified so far. Nevertheless, data convincingly demonstrate that combination therapies (such as dual inhibition of PD-L1 and VEGF) are more effective than the application of single agents. In this review, we will briefly recapitulate the current algorithms for systemic treatment, discuss available results from checkpoint inhibitor trials and give an outlook on future directions of immunotherapy in HCC.

Serum levels of circulating microRNA-107 are elevated in patients with early-stage HCC.

BACKGROUND: Early detection of hepatocellular carcinoma (HCC), the most common primary liver malignancy, is crucial to offer patients a potentially curative treatment strategy such as surgical resection or liver transplantation (LT). However, easily accessible biomarkers facilitating an early diagnosis of HCC as well as a reliable risk prediction are currently missing. The microRNA(miR)-107 has recently been described as a driver of HCC in both murine and human HCC but data on circulating miR-107 in HCC patients are scarce. In the present study, we evaluated a potential diagnostic and/or prognostic role of circulating miR-107 in patients undergoing tumor resection or LT for early-stage HCC. METHODS: The Kmplot bioinformatic tool was used to query publicly available databases (including TCGA, GEO and EGA) in order to analyse the prognostic value of tumoral miR-107 expression in HCC patients (n = 372). Serum levels of miR-107 were measured by qPCR in n = 45 HCC patients undergoing surgical tumor resection (n = 37) or LT (n = 8) as well as n = 18 healthy control samples. Results were correlated with clinical data. RESULTS: A high tumoral expression of miR-107 was associated with a significantly better overall survival compared to patients with low miR-107 expression levels (HR 0.69, 95% CI 0.48-0.99, p = 0.041). In addition, serum levels of miR-107 were significantly higher in HCC patients when compared to healthy controls. However, miR-107 serum levels in HCC patients were independent of different disease etiology, tumor stage or tumor grading. HCC patients with baseline miR-107 expression levels above a calculated ideal prognostic cut-off value (9.82) showed a clear trend towards an impaired overall survival (p = 0.119). CONCLUSION: Tumoral miR-107 expression levels are a potential prognostic marker in early stage HCC. Furthermore, we describe a potential role of circulating miR-107 levels as a diagnostic biomarker in patients with early-stage HCC.

From Liver Cirrhosis to Cancer: The Role of Micro-RNAs in Hepatocarcinogenesis.

In almost all cases, hepatocellular carcinoma (HCC) develops as the endpoint of a sequence that starts with chronic liver injury, progresses to liver cirrhosis, and finally, over years and decades, results in liver cancer. Recently, the role of non-coding RNA such as microRNA (miRNA) has been demonstrated in the context of chronic liver diseases and HCC. Moreover, data from a phase II trial suggested a potential role of microRNAs as therapeutics in hepatitis-C-virus infection, representing a significant risk factor for development of liver cirrhosis and HCC. Despite progress in the clinical management of chronic liver diseases, pharmacological treatment options for patients with liver cirrhosis and/or advanced HCC are still limited. With their potential to regulate whole networks of genes, miRNA might be used as novel therapeutics in these patients but could also serve as biomarkers for improved patient stratification. In this review, we discuss available data on the role of miRNA in the transition from liver cirrhosis to HCC. We highlight opportunities for clinical translation and discuss open issues applicable to future developments.

Immune Regulatory 1 Cells: A Novel and Potent Subset of Human T Regulatory Cells.

A subset of T regulatory cells (Tregs), identified by TIRC7 (T cell immune response cDNA 7) expression is designated as Immune Regulatory 1 Cells (IR1 cells). TIRC7 is an immune checkpoint inhibitor, co-localized with the T- cell receptor, HLA-DR and CTLA-4 during T-cell activation, which delivers regulatory signals via binding to its ligand, HLA-DR α2 domain. IR1 cells express FOXP3, and multiple other markers associated with immune suppression. They constitute as much as 10% of Tregs. IR1 cells strongly inhibit proliferation in mixed lymphocyte reactions, where they express high levels of IL-10. Ex vivo expansion of Tregs over 2 weeks in the presence of an agonist TIRC7 antibody disproportionately expands the IR1 Treg subset, while maintaining high expression of suppressive markers including CD39, IL-10, LAP and GARP. Ex vivo expanded IR1 cells are a potent, homogeneous, stable set of suppressor Tregs with the potential to modulate immune dysregulation. The characteristics of IR1 cells suggest a therapeutic advantage over polyclonal Tregs for therapeutic interventions. Early restoration of immune homeostasis using IR1 cells has the potential to fundamentally alter the natural history of conditions characterized by abnormalities in the T regulatory cell compartment.