The development of flow cytometric biomarkers in human studies and clinical trials has been slowed by inconsistent sample processing, use of cell surface markers, and reporting of immunophenotypes. ...Additionally, the function(s) of distinct cell types as biomarkers cannot be accurately defined without the proper identification of homogeneous populations. As such, we developed a method for the identification and analysis of human leukocyte populations by the use of eight 10-color flow cytometric protocols in combination with novel software analyses. This method utilizes un-manipulated biological sample preparation that allows for the direct quantitation of leukocytes and non-overlapping immunophenotypes. We specifically designed myeloid protocols that enable us to define distinct phenotypes that include mature monocytes, granulocytes, circulating dendritic cells, immature myeloid cells, and myeloid derived suppressor cells (MDSCs). We also identified CD123 as an additional distinguishing marker for the phenotypic characterization of immature LIN-CD33+HLA-DR- MDSCs. Our approach permits the comprehensive analysis of all peripheral blood leukocytes and yields data that is highly amenable for standardization across inter-laboratory comparisons for human studies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
Immunosuppression in glioblastoma (GBM) is an obstacle to effective immunotherapy. GBM-derived immunosuppressive monocytes are central to this. Programmed cell death ligand 1 ...(PD-L1) is an immune checkpoint molecule, expressed by GBM cells and GBM extracellular vesicles (EVs). We sought to determine the role of EV-associated PD-L1 in the formation of immunosuppressive monocytes.
Methods
Monocytes collected from healthy donors were conditioned with GBM-derived EVs to induce the formation of immunosuppressive monocytes, which were quantified via flow cytometry. Donor-matched T cells were subsequently co-cultured with EV-conditioned monocytes in order to assess effects on T-cell proliferation. PD-L1 constitutive overexpression or short hairpin RNA–mediated knockdown was used to determined the role of altered PD-L1 expression.
Results
GBM EVs interact with both T cells and monocytes but do not directly inhibit T-cell activation. However, GBM EVs induce immunosuppressive monocytes, including myeloid-derived suppressor cells (MDSCs) and nonclassical monocytes (NCMs). MDSCs and NCMs inhibit T-cell proliferation in vitro and are found within GBM in situ. EV PD-L1 expression induces NCMs but not MDSCs, and does not affect EV-conditioned monocytes T-cell inhibition.
Conclusion
These findings indicate that GBM EV-mediated immunosuppression occurs through induction of immunosuppressive monocytes rather than direct T-cell inhibition and that, while PD-L1 expression is important for the induction of specific immunosuppressive monocyte populations, immunosuppressive signaling mechanisms through EVs are complex and not limited to PD-L1.
Abstract
Context
Thyroid immune-related adverse events (irAEs) in patients treated with programmed death receptor-1 (PD-1) blockade are increasingly recognized as one of the most common adverse ...effects. Our aim was to determine the incidence and examine the potential mechanisms of anti-PD-1–induced thyroid irAEs.
Design
Single-center, retrospective cohort study.
Patients and Measurements
We studied 93 patients with advanced cancer (ages 24 to 82 years; 60% males) who received at least one infusion of pembrolizumab. Thyroid test results and thyroid imaging modalities were reviewed. Comprehensive 10-color flow cytometry of peripheral blood was performed.
Results
Thirteen (14%) thyroid irAEs were observed. Thyroiditis occurred in seven patients (54%), from which four recovered. New onset of hypothyroidism overt/subclinical developed in three patients. Levothyroxine dosing required doubling in three patients with a known history of hypothyroidism. Thyroperoxidase antibodies were positive in the minority of the patients 4/13 (31%) and diffuse increased 18fludeoxyglucose uptake of the thyroid gland was observed in the majority 7/11 (64%) of patients. We observed more circulating CD56+CD16+ natural killer (NK) cells and an elevated HLA-DR surface expression in the inflammatory intermediate CD14+CD16+ monocytes in anti-PD-1–treated patients.
Conclusions
Thyroid dysfunction is common in cancer patients treated with pembrolizumab. Reversible destructive thyroiditis and overt hypothyroidism are the most common clinical presentations. The mechanism of thyroid destruction appears independent of thyroid autoantibodies and may include T cell, NK cell, and/or monocyte-mediated pathways. Because the thyroid is a frequent target of anti-PD-1 therapies, patients with therapeutically refractory thyroid cancer may be ideal candidates for this treatment.
We studied 93 pembrolizumab-treated patients and found that destructive thyroiditis was the most common clinical presentation, possibly triggered by T cell, NK cell, and/or monocyte-mediated pathways.
Atherosclerotic renovascular disease (RVD) reduces renal blood flow (RBF) and GFR and accelerates poststenotic kidney (STK) tissue injury. Preclinical studies indicate that mesenchymal stem cells ...(MSCs) can stimulate angiogenesis and modify immune function in experimental RVD. We assessed the safety and efficacy of adding intra-arterial autologous adipose-derived MSCs into STK to standardized medical treatment in human subjects without revascularization. The intervention group (
=14) received a single infusion of MSC (1.0 × 10
or 2.5 × 10
cells/kg;
=7 each) plus standardized medical treatment; the medical treatment only group (
=14) included subjects matched for age, kidney function, and stenosis severity. We measured cortical and medullary volumes, perfusion, and RBF using multidetector computed tomography. We assessed tissue oxygenation by blood oxygen level-dependent MRI and GFR by iothalamate clearance. MSC infusions were well tolerated. Three months after infusion, cortical perfusion and RBF rose in the STK (151.8-185.5 ml/min,
=0.01); contralateral kidney RBF increased (212.7-271.8 ml/min,
=0.01); and STK renal hypoxia (percentage of the whole kidney with R2*>30/s) decreased (12.1% interquartile range, 3.3%-17.8% to 6.8% interquartile range, 1.8%-12.9%,
=0.04). No changes in RBF occurred in medical treatment only subjects. Single-kidney GFR remained stable after MSC but fell in the medical treatment only group (-3% versus -24%,
=0.04). This first-in-man dose-escalation study provides evidence of safety of intra-arterial infusion of autologous MSCs in patients with RVD. MSC infusion without main renal artery revascularization associated with increased renal tissue oxygenation and cortical blood flow.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a median lifespan of 2-3 years after diagnosis. There are few meaningful treatments that alter progression in this ...disease. Preclinical and clinical studies have demonstrated that neuroinflammation may play a key role in the progression rate of ALS. Despite this, there are no validated biomarkers of neuroinflammation for use in clinical practice or clinical trials. Biomarkers of neuroinflammation could improve patient management, provide new therapeutic targets, and possibly help stratify clinical trial selection and monitoring. However, attempts to identify a singular cause of neuroinflammation have not been successful. Here, we performed multi-parameter flow cytometry to comprehensively assess 116 leukocyte populations and phenotypes from lymphocytes, monocytes, and granulocytes in a cohort of 80 ALS patients. We identified 32 leukocyte phenotypes that were altered in ALS patients compared to age and gender matched healthy volunteers (HV) that included phenotypes of both inflammation and immune suppression. Unsupervised hierarchical clustering and principle component analysis of ALS and HV immunophenotypes revealed two distinct immune profiles of ALS patients. ALS patients were clustered into a profile distinct from HVs primarily due to differences in a multiple T cell phenotypes, CD3+CD56+ T cells and HLA-DR on monocytes. Patients clustered into an abnormal immune profile were younger, more likely to have a familial form of the disease, and survived longer than those patients who clustered similarly with healthy volunteers (344 weeks versus 184 weeks; p = 0.012). The data set generated from this study establishes an extensive accounting of immunophenotypic changes readily suitable for biomarker validation studies. The extensive immune system changes measured in this study indicate that normal immune homeostatic mechanisms are disrupted in ALS patients, and that multiple immune states likely exist within a population of patients with ALS.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cell therapy is an emerging field that uses cells as living drugs to treat a broad array of acute and chronic diseases. Most cell therapies in clinical trials are made using standard bench methods, ...whose open processing require manufacturing in expensive GMP cleanrooms. As cell therapies progress, new methods are needed to enable scalable manufacturing while maintaining process integrity, reducing environmental exposure, and limiting critical cell and reagent use. Here, we introduce capture and reagent exchange (CARE) wells that allow critical processing steps to be integrated into a closed microfluidic device. The unique property of CARE wells is that they allow reagent exchange from an attached channel without cell loss from wells. We show through simulation and experiment that this feature is present in cylindrical wells whose depth is sufficient to generate multiple recirculating vortices and is independent of flow rate in the channel. We demonstrate that CARE wells can be used to perform cell separation, on-chip labeling, and characterization of monocytes as the first steps toward a closed microfluidic system for production of dendritic cell therapies. Immunomagnetic separation of CD14 + monocytes from peripheral blood mononuclear cells (PBMCs) into wells was performed with purity of 97 ± 2% and capture efficiency of 50 ± 17%. On-chip labeling, washing, and characterization were performed using two cell surface markers (CD14 and HLA-DR) on over 3000 cells captured in a 5193-well device. The combination of high purity separation and reagent exchange without cell loss with robust performance over wide range of input and operating conditions makes this technique a promising approach for scalable manufacturing and analysis of cell therapies.
Spinal cord injury (SCI) is a devastating condition with limited pharmacological treatment options to restore function. Regenerative approaches have recently attracted interest as an adjuvant to ...current standard of care. Adipose tissue-derived (AD) mesenchymal stem cells (MSCs) represent a readily accessible cell source with high proliferative capacity. The CELLTOP study, an ongoing multidisciplinary phase 1 clinical trial conducted at Mayo Clinic (ClinicalTrials.gov Identifier: NCT03308565), is investigating the safety and efficacy of intrathecal autologous AD-MSCs in patients with blunt, traumatic SCI. In this initial report, we describe the outcome of the first treated patient, a 53-year-old survivor of a surfing accident who sustained a high cervical American Spinal Injury Association Impairment Scale grade A SCI with subsequent neurologic improvement that plateaued within 6 months following injury. Although he improved to an American Spinal Injury Association grade C impairement classification, the individual continued to be wheelchair bound and severely debilitated. After study enrollment, an adipose tissue biopsy was performed and MSCs were isolated, expanded, and cryopreserved. Per protocol, the patient received an intrathecal injection of 100 million autologous AD-MSCs infused after a standard lumbar puncture at the L3-4 level 11 months after the injury. The patient tolerated the procedure well and did not experience any severe adverse events. Clinical signs of efficacy were observed at 3, 6, 12, and 18 months following the injection in both motor and sensory scores based on International Standards for Neurological Classification of Spinal Cord Injury. Thus, in this treated individual with SCI, intrathecal administration of AD-MSCs was feasible and safe and suggested meaningful signs of improved, rather than stabilized, neurologic status warranting further clinical evaluation.
Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained ...underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.
Osteogenic differentiation is initiated by transcriptional and post-transcriptional epigenetic mechanisms.
Inhibition of H3K27 methyltransferase EZH2 enhances osteogenic commitment of human mesenchymal progenitors, and its depletion in mouse mesenchymal cells causes multiple skeletal abnormalities.
EZH2 is required for skeletal patterning and bone formation.
EZH2-dependent epigenetic mechanisms control osteogenesis both in vitro and in vivo.
Immunosuppression is a known risk factor for B-cell non-Hodgkin lymphoma (NHL), yet mechanisms of tumor-associated immunosuppression remain to be fully characterized. We examined the immunophenotype ...of 40 NHL patients and 27 age-matched healthy volunteers to better understand systemic immune suppression. NHL peripheral blood mononuclear cells had significantly decreased interferon-γ production and proliferation. This suppression was not the result of regulatory T cells, interleukin-6 or interleukin-10, as these factors were not different between NHL and healthy volunteers (controls). We were able to restore T-cell proliferation by removing NHL monocytes, suggesting that these monocytes are suppressive. This suppression was mediated in part through arginine metabolism as exogenous arginine supplementation partially overcame monocytes' suppression of T-cell proliferation in vitro and NHL patients had elevated arginase I in their plasma. NHL monocytes had impaired STAT1 phosphorylation and interferon-α production to CpG stimulation and a dendritic cell differentiation deficiency. Further studies demonstrated that monocytes from NHL patients had decreased HLA-DR and Tumor necrosis factor-α receptor II (CD120b) expression compared with controls (CD14+HLA-DRlow/−CD120blow). Patients with increased ratios of CD14+HLA-DRlow/− monocytes had more aggressive disease and suppressed immune functions. In summary, we report that CD14+HLA-DRlow/− monocytes are a major and multifactorial contributor to systemic immunosuppression in NHL.
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