DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance.

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

Postoperative cellular stress in the kidney is associated with an early systemic γδ T-cell immune cell response.

BACKGROUND: Basic science data suggest that acute kidney injury (AKI) induced by ischemia-reperfusion injury (IRI) is an inflammatory process involving the adaptive immune response. Little is known about the T-cell contribution in the very early phase, so we investigated if tubular cellular stress expressed by elevated cell cycle biomarkers is associated with early changes in circulating T-cell subsets, applying a bedside-to-bench approach. METHODS: Our observational pilot study included 20 consecutive patients undergoing endovascular aortic repair for aortic aneurysms affecting the renal arteries, thereby requiring brief kidney hypoperfusion and reperfusion. Clinical-grade flow cytometry-based immune monitoring of peripheral immune cell populations was conducted perioperatively and linked to tubular cell stress biomarkers ([TIMP-2]•[IGFBP7]) immediately after surgery. To confirm clinical results and prove T-cell infiltration in the kidney, we simulated tubular cellular injury in an established mouse model of mild renal IRI. RESULTS: A significant correlation between tubular cell injury and a peripheral decline of γδ T cells, but no other T-cell subpopulation, was discovered within the first 24 hours (r = 0.53; p = 0.022). Turning to a mouse model of kidney warm IRI, a similar decrease in circulating γδ T cells was found and concomitantly was associated with a 6.65-fold increase in γδ T cells (p = 0.002) in the kidney tissue without alterations in other T-cell subsets, consistent with our human data. In search of a mechanistic driver of IRI, we found that the damage-associated molecule high-mobility group box 1 protein HMGB1 was significantly elevated in the peripheral blood of clinical study subjects after tubular cell injury (p = 0.019). Correspondingly, HMGB1 RNA content was significantly elevated in the murine kidney. CONCLUSIONS: Our investigation supports a hypothesis that γδ T cells are important in the very early phase of human AKI and should be considered when designing clinical trials aimed at preventing kidney damage. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01915446 . Registered on 5 Aug 2013.

Novel molecules mediate specialized functions of human regulatory macrophages.

PURPOSE OF REVIEW: Now that adoptive transfer of regulatory macrophages (Mregs) is clinically practicable, we ask whether this approach could be used to achieve self-sustaining peripheral regulation and what mechanisms may be involved. RECENT FINDINGS: Dehydrogenase/reductase 9 (DHRS9)-expressing Mregs are a specialized subset of monocyte-derived macrophages that are currently being investigated as a tolerogenic cell-based therapy. Human Mregs are defined by their capacity to convert naïve CD4 T cells to IL-10-secreting FoxP3 regulatory T cells (Tregs) through an activation-dependent process involving signals mediated by TGF-ß, retinoic acid, indoleamine 2,3-dioxygenase activity, notch and progestagen associated endometrial protein (PAEP). Mreg-induced iTregs (miTregs) are a phenotypically distinct type of in-vitro-derived human iTreg that expresses butyrophilin-like protein 8 (BTNL8) and T cell immunoreceptor with Ig and ITIM domains (TIGIT). miTregs are nonspecifically suppressive of mitogen-stimulated bystander T cell proliferation and inhibit TNFα-induced maturation of monocyte-derived dendritic cells. Preclinical and clinical studies find that intravenous infusion of allogeneic Mregs leads to enrichment of circulating TIGIT Tregs. SUMMARY: These results suggest a feed-forward mechanism by which Mreg treatment could promote solid organ transplant acceptance through rapid induction of direct pathway Tregs.

Laser ablation-inductively coupled plasma mass spectrometry: an emerging technology for detecting rare cells in tissue sections.

Administering immunoregulatory cells to patients as medicinal agents is a potentially revolutionary approach to the treatment of immunologically mediated diseases. Presently, there are no satisfactory, clinically applicable methods of tracking human cells in patients with adequate spatial resolution and target cell specificity over a sufficient period of time. Laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS) represents a potential solution to the problem of detecting very rare cells in tissues. In this article, this exquisitely sensitive technique is applied to the tracking of gold-labeled human regulatory macrophages (Mregs) in immunodeficient mice. Optimal conditions for labeling Mregs with 50-nm gold particles were investigated by exposing Mregs in culture to variable concentrations of label: Mregs incubated with 3.5 × 10(9) particles/ml for 1 h incorporated an average of 3.39 × 10(8) Au atoms/cell without loss of cell viability. Analysis of single, gold-labeled Mregs by LA-ICP-MS registered an average of 1.9 × 10(5) counts/cell. Under these conditions, 100% labeling efficiency was achieved, and label was retained by Mregs for ≥36 h. Gold-labeled Mregs adhered to glass surfaces; after 24 h of culture, it was possible to colabel these cells with human-specific (154)Sm-tagged anti-HLA-DR or (174)Yb-tagged anti-CD45 mAbs. Following injection into immunodeficient mice, signals from gold-labeled human Mregs could be detected in mouse lung, liver, and spleen for at least 7 d by solution-based inductively coupled plasma mass spectrometry and LA-ICP-MS. These promising results indicate that LA-ICP-MS tissue imaging has great potential as an analytical technique in immunology.

Clinical management of patients receiving cell-based immunoregulatory therapy.

Administering immunoregulatory cells as medicinal agents is a revolutionary approach to the treatment of immunologically mediated diseases. Isolating, propagating, and modifying cells before applying them to patients allows complementation of specific cellular functions, which opens astonishing new possibilities for gain-of-function antigen-specific treatments in autoimmunity, chronic inflammatory disorders, and transplantation. This critical review presents a systematic assessment of the potential clinical risks posed by cell-based immunotherapy, focusing on treatment of renal transplant recipients with regulatory macrophages as a concrete example.

IFN-γ-induced iNOS expression in mouse regulatory macrophages prolongs allograft survival in fully immunocompetent recipients.

Mouse monocytes exposed to macrophage colony-stimulating factor (M-CSF) and interferon-γ (IFN-γ) were driven to a novel suppressor phenotype. These regulatory macrophages (M regs) expressed markers distinguishing them from M0-, M1-, and M2-polarized macrophages and monocyte-derived dendritic cells (DCs). M regs completely suppressed polyclonal T cell proliferation through an inducible nitric oxide synthase (iNOS)-dependent mechanism. Additionally, M regs eliminated cocultured T cells in an allospecific fashion. In a heterotopic heart transplant model, a single intravenous administration of 5 × 10(6) donor-strain M regs before transplantation significantly prolonged allograft survival in fully immunocompetent recipients using both the stringent C3H-to-BALB/c (32.6 ± 4.5 versus 8.7 ± 0.2 days) and B6-to-BALB/c (31.1 ± 12 versus 9.7 ± 0.4 days) strain combinations. Nos2-deficient M regs did not prolong allograft survival, proving that M reg function in vivo is iNOS-dependent and mediated by living cells. M regs were detectable for at least 2 weeks postinfusion in allogeneic recipients. In their origin, development, phenotypic relationship with other in vitro-derived macrophages and functions, there are solid grounds to assert a near-equivalence of mouse and human M regs. It is concluded that mouse M regs represent a novel, phenotypically distinct subset of suppressor macrophages. Clinical applications of M reg therapy as an adjunct immunosuppressive therapy are currently being investigated within The ONE Study.

Restricting datasets to classifiable samples augments discovery of immune disease biomarkers.

Immunological diseases are typically heterogeneous in clinical presentation, severity and response to therapy. Biomarkers of immune diseases often reflect this variability, especially compared to their regulated behaviour in health. This leads to a common difficulty that frustrates biomarker discovery and interpretation - namely, unequal dispersion of immune disease biomarker expression between patient classes necessarily limits a biomarker's informative range. To solve this problem, we introduce dataset restriction, a procedure that splits datasets into classifiable and unclassifiable samples. Applied to synthetic flow cytometry data, restriction identifies biomarkers that are otherwise disregarded. In advanced melanoma, restriction finds biomarkers of immune-related adverse event risk after immunotherapy and enables us to build multivariate models that accurately predict immunotherapy-related hepatitis. Hence, dataset restriction augments discovery of immune disease biomarkers, increases predictive certainty for classifiable samples and improves multivariate models incorporating biomarkers with a limited informative range. This principle can be directly extended to any classification task.

Soluble CD46 as a diagnostic marker of hepatic steatosis.

BACKGROUND: The increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) incurs substantial morbidity, mortality and healthcare costs. Detection and clinical intervention at early stages of disease improves prognosis; however, we are currently limited by a lack of reliable diagnostic tests for population screening and monitoring responses to therapy. To address this unmet need, we investigated human invariant Natural Killer T cell (iNKT) activation by fat-loaded hepatocytes, leading to the discovery that circulating soluble CD46 (sCD46) levels accurately predict hepatic steatosis. METHODS: sCD46 in plasma was measured using a newly developed immuno-competition assay in two independent cohorts: Prospective living liver donors (n = 156; male = 66, female = 90) and patients with liver tumours (n = 91; male = 58, female = 33). sCD46 levels were statistically evaluated as a predictor of hepatic steatosis. FINDINGS: Interleukin-4-secreting (IL-4+) iNKT cells were over-represented amongst intrahepatic lymphocytes isolated from resected human liver samples. IL-4+ iNKT cells preferentially developed in cocultures with a fat-loaded, hepatocyte-like cell line, HepaRG. This was attributed to induction of matrix metalloproteases (MMP) in fat-loaded HepaRG cells and primary human liver organoids, which led to indiscriminate cleavage of immune receptors. Loss of cell-surface CD46 resulted in unrepressed differentiation of IL-4+ iNKT cells. sCD46 levels were elevated in patients with hepatic steatosis. Discriminatory cut-off values for plasma sCD46 were found that accurately classified patients according to histological steatosis grade. INTERPRETATION: sCD46 is a reliable clinical marker of hepatic steatosis, which can be conveniently and non-invasively measured in serum and plasma samples, raising the possibility of using sCD46 levels as a diagnostic method for detecting or grading hepatic steatosis. FUNDING: F.B. was supported by the Else Kröner Foundation (Award 2016_kolleg.14). G.G. was supported by the Bristol Myers Squibb Foundation for Immuno-Oncology (Award FA-19-009). N.S. was supported by a Wellcome Trust Fellowship (211113/A/18/Z). J.A.H. received funding from the European Union's Horizon 2020 research and innovation programme (Award 860003). J.M.W. received funding from the Else Kröner Foundation (Award 2015_A10).

Cutting Edge: Immunological consequences and trafficking of human regulatory macrophages administered to renal transplant recipients.

Regulatory macrophages (M regs) were administered to two living-donor renal transplant recipients. Both patients were minimized to low-dose tacrolimus monotherapy within 24 wk of transplantation and subsequently maintained excellent graft function. After central venous administration, most M regs remained viable and were seen to traffic from the pulmonary vasculature via the blood to liver, spleen, and bone marrow. By 1 y posttransplantation, both patients displayed patterns of peripheral blood gene expression converging upon the IOT-RISET signature. Furthermore, both patients maintained levels of peripheral blood FOXP3 and TOAG-1 mRNA expression within the range consistent with nonrejection. It is concluded that M regs warrant further study as a potential immune-conditioning therapy for use in solid-organ transplantation. The results of this work are being used to inform the design of The ONE Study, a multinational clinical trial of immunomodulatory cell therapy in renal transplantation.

Human regulatory macrophages as a cell-based medicinal product.

PURPOSE OF REVIEW: To consolidate our basic scientific and technological appreciation of human regulatory macrophages (M reg) as a cell-based medicinal product for use as an adjunct immunosuppressive therapy in organ transplantation. RECENT FINDINGS: Building on the original observation that crude preparations of IFN-γ-stimulated allogeneic macrophages prolong allograft survival in experimental animals, we have arrived at a detailed understanding of the derivation, phenotype and T-cell-suppressive potential of a population of in-vitro-derived human macrophages, which have been designated M regs. This basic scientific knowledge has inspired methodological advances in M reg manufacture, leading to a purer and more homogeneous cell product. In turn, cells produced by these improved protocols have been applied in the clinic, so completing a cycle of technological development. Studying the migration and physiological impact of M reg administration in patients provides a measure of reassurance that the procedure is well tolerated. Cutting-edge strategies to assess the immunological status of solid organ transplant recipients allow the biological effects of M reg treatment to be monitored. SUMMARY: A view of the human M reg as a novel, stringently defined medicinal product is presented, opening exciting possibilities for its future investigation as a therapy in solid organ transplantation and beyond.

Regulatory macrophages as therapeutic targets and therapeutic agents in solid organ transplantation.

PURPOSE OF REVIEW: This review aims to provide a basic introduction to human macrophage biology and an appreciation of the diverse roles played by macrophage subsets in allograft damage and repair. Current and future strategies for therapeutically manipulating macrophage behaviour are discussed. RECENT FINDINGS: Macrophages are extremely versatile effector cells that exert both immunostimulatory and immunosuppressive effects. This adaptability cannot be explained by differentiation into committed sublineages, but instead reflects the ability of macrophages to rapidly transition between states of functional polarisation. Consequently, categorisation of macrophage subpopulations is not straightforward and this, in turn, creates difficulties in studying their pathophysiology. Nevertheless, particular macrophage subpopulations have been implicated in exacerbating or attenuating ischaemia-reperfusion injury, rejection reactions and allograft fibrosis. Three general strategies for therapeutically targeting macrophages can be envisaged, namely, depletional approaches, in-situ repolarisation towards a regulatory or tissue-reparative phenotype, and ex-vivo generation of regulatory macrophages (M reg) as a cell-based therapy. SUMMARY: As critical determinants of the local and systemic immune response to solid organ allografts, macrophage subpopulations represent attractive therapeutic targets. Rapid progress is being made in the implementation of novel macrophage-targeted therapies, particularly in the use of ex-vivo-generated M regs as a cell-based medicinal product.

External validation of biomarkers for immune-related adverse events after immune checkpoint inhibition.

Immune checkpoint inhibitors have revolutionized treatment of advanced melanoma, but commonly cause serious immune-mediated complications. The clinical ambition of reserving more aggressive therapies for patients least likely to experience immune-related adverse events (irAE) has driven an extensive search for predictive biomarkers. Here, we externally validate the performance of 59 previously reported markers of irAE risk in a new cohort of 110 patients receiving Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA-4) therapy. Alone or combined, the discriminatory value of these routine clinical parameters and flow cytometry biomarkers was poor. Unsupervised clustering of flow cytometry data returned four T cell subsets with higher discriminatory capacity for colitis than previously reported populations, but they cannot be considered as reliable classifiers. Although mechanisms predisposing some patients to particular irAEs have been described, we are presently unable to capture adequate information from pre-therapy flow cytometry and clinical data to reliably predict risk of irAE in most cases.

Development of a Flow Cytometry Assay to Predict Immune Checkpoint Blockade-Related Complications.

Treatment of advanced melanoma with combined immune checkpoint inhibitor (ICI) therapy is complicated in up to 50% of cases by immune-related adverse events (irAE) that commonly include hepatitis, colitis and skin reactions. We previously reported that pre-therapy expansion of cytomegalovirus (CMV)-reactive CD4+ effector memory T cells (TEM) predicts ICI-related hepatitis in a subset of patients with Stage IV melanoma given αPD-1 and αCTLA-4. Here, we develop and validate a 10-color flow cytometry panel for reliably quantifying CD4+ TEM cells and other biomarkers of irAE risk in peripheral blood samples. Compared to previous methods, our new panel performs equally well in measuring CD4+ TEM cells (agreement = 98%) and is superior in resolving CD4+ CD197+ CD45RA- central memory T cells (TCM) from CD4+ CD197+ CD45RA+ naive T cells (Tnaive). It also enables us to precisely quantify CD14+ monocytes (CV = 6.6%). Our new "monocyte and T cell" (MoT) assay predicts immune-related hepatitis with a positive predictive value (PPV) of 83% and negative predictive value (NPV) of 80%. Our essential improvements open the possibility of sharing our predictive methods with other clinical centers. Furthermore, condensing measurements of monocyte and memory T cell subsets into a single assay simplifies our workflows and facilitates computational analyses.

Virus-specific memory T cell responses unmasked by immune checkpoint blockade cause hepatitis.

Treatment of advanced melanoma with combined PD-1/CTLA-4 blockade commonly causes serious immune-mediated complications. Here, we identify a subset of patients predisposed to immune checkpoint blockade-related hepatitis who are distinguished by chronic expansion of effector memory CD4+ T cells (TEM cells). Pre-therapy CD4+ TEM cell expansion occurs primarily during autumn or winter in patients with metastatic disease and high cytomegalovirus (CMV)-specific serum antibody titres. These clinical features implicate metastasis-dependent, compartmentalised CMV reactivation as the cause of CD4+ TEM expansion. Pre-therapy CD4+ TEM expansion predicts hepatitis in CMV-seropositive patients, opening possibilities for avoidance or prevention. 3 of 4 patients with pre-treatment CD4+ TEM expansion who received αPD-1 monotherapy instead of αPD-1/αCTLA-4 therapy remained hepatitis-free. 4 of 4 patients with baseline CD4+ TEM expansion given prophylactic valganciclovir and αPD-1/αCTLA-4 therapy remained hepatitis-free. Our findings exemplify how pathogen exposure can shape clinical reactions after cancer therapy and how this insight leads to therapeutic innovations.

A refined characterisation of the NeoHepatocyte phenotype necessitates a reappraisal of the transdifferentiation hypothesis.

Under certain culture conditions human peripheral blood monocytes may be induced to express phenotypic markers of non-haematopoietic lineages, including hepatocyte-defining traits. One such example, the NeoHepatocyte, was previously shown to express a broad panel of hepatocyte-like marker antigens and metabolic activities, both in vitro and following engraftment in the liver of immunodeficient mice. In this report, a refined description of NeoHepatocytes, with regard to their expression of xenobiotic-metabolising enzymes, morphology, hepatocyte marker expression and cell surface phenotype, is presented in comparison with human macrophages in defined states of activation. Contrary to prior assertions, it would seem more likely that NeoHepatocytes express particular hepatocyte-defining genes during a normal programme of macrophage differentiation rather than undergoing a process of transdifferentiation to become hepatocyte-like cells.

Myeloid-Derived Suppressor Cells in Kidney Transplant Recipients and the Effect of Maintenance Immunotherapy.

Myeloid-derived suppressor cells (MDSC) represent a heterogeneous group of myeloid regulatory cells that were originally described in cancer. Several studies in animal models point to MDSC as important players in the induction of allograft tolerance due to their immune modulatory function. Most of the published studies have been performed in animal models, and the data addressing MDSCs in human organ transplantation are scarce. We evaluated the phenotype and function of different MDSCs subsets in 38 kidney transplant recipients (KTRs) at different time points. Our data indicate that monocytic MDSCs (Mo-MDSC) increase in KTR at 6 and 12 months posttransplantation. On the contrary, the percentages of polymorphonuclear MDSC (PMN-MDSC) and early-stage MDSC (e-MDSC) are not significantly increased. We evaluated the immunosuppressive activity of Mo-MDSC in KTR and confirmed their ability to increase regulatory T cells (Treg) in vitro. Interestingly, when we compared the ability of Mo-MDSC to suppress T cell proliferation, we observed that tacrolimus, but not rapamycin-treated KTR, was able to inhibit CD4+ T cell proliferation in vitro. This indicates that, although mTOR inhibitors are widely regarded as supportive of regulatory responses, rapamycin may impair Mo-MDSC function, and suggests that the choice of immunosuppressive therapy may determine the tolerogenic pathway and participating immune cells that promote organ transplant acceptance in KTR.

Administration of donor-derived transplant acceptance-inducing cells to the recipients of renal transplants from deceased donors is technically feasible.

Patient KW, a 36-yr-old male renal transplant recipient, received transplant acceptance-inducing cells (TAICs) as an adjunct immunosuppressive therapy. In the weeks post-transplantation, the patient's conventional immunosuppressive treatment was gradually minimized, such that, from the 21st wk post-transplantation onwards, the patient was stably maintained on tacrolimus monotherapy. Treatment with TAICs was without complication, both at the time of administration and in the four-yr follow-up period. It is concluded that the production and administration of TAICs to recipients of kidney transplants from deceased donors is technically feasible.

Predicting Early Viral Control under Direct-Acting Antiviral Therapy for Chronic Hepatitis C Virus Using Pretreatment Immunological Markers.

Recent introduction of all-oral direct-acting antiviral (DAA) treatment has revolutionized care of patients with chronic hepatitis C virus (HCV) infection. Regrettably, the high cost of DAA treatment is burdensome for healthcare systems and may be prohibitive for some patients who would otherwise benefit. Understanding how patient-related factors influence individual responses to DAA treatment may lead to more efficient prescribing. In this observational study, patients with chronic HCV infection were comprehensively monitored by flow cytometry to identify pretreatment immunological variables that predicted HCV RNA negativity within 4 weeks of commencing DAA treatment. Twenty-three patients [genotype 1a (n = 10), 1b (n = 9), and 3 (n = 4)] were treated with daclatasvir plus sofosbuvir (SOF) (n = 15), ledipasvir plus SOF (n = 4), or ritonavir-boosted paritaprevir, ombitasvir, and dasabuvir (n = 4). DAA treatment most prominently altered the distribution of CD8+ memory T cell subsets. Knowing only pretreatment frequencies of CD3+ and naive CD8+ T cells allowed correct classification of 83% of patients as "fast" (HCV RNA-negative by 4 weeks) or "slow" responders. In a prospective cohort, these parameters correctly classified 90% of patients. Slow responders exhibited higher frequencies of CD3+ T cells, CD8+ TEM cells, and CD5high CD27- CD57+ CD8+ chronically activated T cells, which is attributed to bystander hyperactivation of virus-non-specific CD8+ T cells. Taken together, non-specific, systemic CD8+ T cell activation predicted a longer time to viral clearance. This discovery allows pretreatment identification of individuals who may not require a full 12-week course of DAA therapy; in turn, this could lead to individualized prescribing and more efficient resource allocation.

TIGIT+ iTregs elicited by human regulatory macrophages control T cell immunity.

Human regulatory macrophages (Mreg) have shown early clinical promise as a cell-based adjunct immunosuppressive therapy in solid organ transplantation. It is hypothesised that recipient CD4+ T cell responses are actively regulated through direct allorecognition of donor-derived Mregs. Here we show that human Mregs convert allogeneic CD4+ T cells to IL-10-producing, TIGIT+ FoxP3+-induced regulatory T cells that non-specifically suppress bystander T cells and inhibit dendritic cell maturation. Differentiation of Mreg-induced Tregs relies on multiple non-redundant mechanisms that are not exclusive to interaction of Mregs and T cells, including signals mediated by indoleamine 2,3-dioxygenase, TGF-ß, retinoic acid, Notch and progestagen-associated endometrial protein. Preoperative administration of donor-derived Mregs to living-donor kidney transplant recipients results in an acute increase in circulating TIGIT+ Tregs. These results suggest a feed-forward mechanism by which Mreg treatment promotes allograft acceptance through rapid induction of direct-pathway Tregs.