Immune checkpoint inhibitor (ICI) use remains a challenge in patients with solid organ allografts as most would undergo rejection. In a melanoma patient in whom programmed-death 1 (PD-1) blockade ...resulted in organ rejection and colitis, the addition of the mTOR inhibitor sirolimus resulted in ongoing anti-tumor efficacy while promoting allograft tolerance. Strong granzyme B
, interferon (IFN)-γ
CD8
cytotoxic T cell and circulating regulatory T (T
) cell responses were noted during allograft rejection, along with significant eosinophilia and elevated serum IL-5 and eotaxin levels. Co-treatment with sirolimus abated cytotoxic T cell numbers and eosinophilia, while elevated T
cell numbers in the peripheral blood were maintained. Interestingly, numbers of IFN-γ
CD4
T cells and serum IFN-γ levels increased with the addition of sirolimus treatment likely promoting ongoing anti-PD-1 efficacy. Thus, our results indicate that sirolimus has the potential to uncouple anti-PD-1 therapy toxicity and efficacy.
Programmed cell death 1 (PD-1) is an inhibitory receptor involved in T-cell activation, tolerance and exhaustion. Little is known on how the expression of PD-1 is controlled during T-cell activation. ...Recent studies demonstrated that NFATc1 and IRF9 regulate Pdcd1 (PD-1) transcription and that T-bet acts as a transcriptional repressor. In this study, we have investigated the role of the Notch signaling pathway in PD-1 regulation. Using specific inhibitors of the Notch signaling pathway, we showed decreased PD-1 expression and inhibition of Pdcd1 transcription by activated CD8(+) T cells. Chromatin immunoprecipitation further showed occupancy of the Pdcd1 promoter with RBPJk and Notch1 intracellular domain at RBPJk-binding sites. Our results identify the Notch signaling pathway as an important regulator of PD-1 expression by activated CD8(+) T cells.
Transplant vasculopathy is characterized by endothelial apoptosis, which modulates the local microenvironment. Milk fat globule epidermal growth factor 8 (MFG-E8), which is released by apoptotic ...endothelial cells, limits tissue damage and inflammation by promoting anti-inflammatory macrophages. We aimed to study its role in transplant vasculopathy using the murine aortic allotransplantation model. BALB/c mice were transplanted with fully mismatched aortic transplants from MFG-E8 knockout (KO) or wild type (WT) C57BL/6J mice. Thereafter, mice received MFG-E8 (or vehicle) injections for 9 weeks prior to histopathological analysis of allografts for intimal proliferation (hematoxylin and eosin staining) and leukocyte infiltration assessment (immunofluorescence). Phenotypes of blood leukocytes and humoral responses were also evaluated (flow cytometry and ELISA). Mice receiving MFG-E8 KO aortas without MFG-E8 injections had the most severe intimal proliferation (p < 0.001). Administration of MFG-E8 decreased intimal proliferation, especially in mice receiving MFG-E8 KO aortas. Administration of MFG-E8 also increased the proportion of anti-inflammatory macrophages among graft-infiltrating macrophages (p = 0.003) and decreased systemic CD4+ and CD8+ T-cell activation (p < 0.001). An increase in regulatory T cells occurred in both groups of mice receiving WT aortas (p < 0.01). Thus, the analarmin MFG-E8 appears to be an important protein for reducing intimal proliferation in this murine model of transplant vasculopathy. MFG-E8 effects are associated with intra-allograft macrophage reprogramming and systemic T-cell activation dampening.
Adoptive cell therapy (ACT) shows success against treatment-resistant cancers, but is limited by the large number of intravenously delivered T cells required and toxicity related to systemic ...administration. In this work, we hypothesized that localized T cell delivery in an
gelling chitosan hydrogel will allow similar treatment efficacy despite delivering fewer cells than systemic intravenous delivery. A rapidly gelling chitosan gel with good mechanical properties was used for this study. Gel biocompatibility and biodegradability were tested over 8 weeks in mice. No adverse effects were observed. The gel elicited a local granulomatous reaction (foreign body reaction), degrading by about 75% volume at 8 weeks. The survival, escape and bioactivity against the tumour cells of encapsulated murine lymphocytes (OT-I) and human Jurkat cells were confirmed
by live/dead assay and flow cytometry. Efficacy was studied using a mouse tumour model where the injected OT-I can specifically recognize and attack ovalbumin (OVA) protein-expressing tumours. The OT-I cell delivery scaffold was compared to untreated controls, OT-I in saline and intravenous systemic treatment with 3-fold more OT-I, observing tumour growth and localization by intravital microscopy and histology. Gel-encapsulated OT-I limited tumour growth significantly up to 11 days after treatment compared to that of untreated mice and mice with longer PBS-suspended OT-I treatment (9 days), but slightly less than that of mice with IV-delivered OT-I treatment (14 days). No significant difference was observed when directly comparing the gel and IV treatments. Although further optimization of the treatment is required, this work shows the feasibility and potential of the chitosan gel for localised OT-I delivery in cancer immunotherapy.
BackgroundHydrolysis of extracellular ATP to adenosine (eADO) is an important immune checkpoint in cancer immunology. We here investigated the impact of the eADO pathway in high-grade serous ovarian ...cancer (HGSC) using multiparametric platforms.MethodsWe performed a transcriptomic meta-analysis of eADO-producing CD39 and CD73, an eADO signaling gene signature, immune gene signatures and clinical outcomes in approximately 1200 patients with HGSC. Protein expression, localization and prognostic impact of CD39, CD73 and CD8 were then performed on approximately 1000 cases on tissue microarray, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry and single-cell RNA sequencing on a subset of patients.ResultsConcomitant CD39 and CD73 gene expression, as well as high levels of an eADO gene signature, were associated with worse prognosis in patients with HGSC, notably in the immunoregulatory molecular subtype, characterized by an immune-active microenvironment. CD39 was further associated with primary chemorefractory and chemoresistant human HGSC and platinum-based chemotherapy of murine HGSC was significantly more effective in CD39-deficient mice. At protein level, CD39 and CD73 were predominantly expressed by cancer-associated fibroblasts, and CD39 was expressed on severely exhausted, clonally expanded and putative tissue-resident memory TILs.ConclusionsOur study revealed the clinical, immunological, subtype-specific impacts of eADO signaling in HGSC, unveiled the chemoprotective effect of CD39 and supports the evaluation of eADO-targeting agents in patients with ovarian cancer.
Aneurysmal subarachnoid hemorrhage (SAH) is a catastrophic disease with devastating consequences, including a high mortality rate and severe disabilities among survivors. Inflammation is induced ...following SAH, but the exact role and phenotype of innate immune cells remain poorly characterized. We investigated the inflammatory components of the early brain injury in an animal model and in SAH patients.
SAH was induced through injection of blood in the subarachnoid space of C57Bl/6 J wild-type mice. Prospective blood collections were obtained at 12 h, days 1, 2, and 7 to evaluate the systemic inflammatory consequences of SAH by flow cytometry and enzyme-linked immunosorbent-assay (ELISA). Brains were collected, enzymatically digested, or fixed to characterize infiltrating inflammatory cells and neuronal death using flow cytometry and immunofluorescence. Phenotypic evaluation was performed at day 7 using the holding time and footprint tests. We then compared the identified inflammatory proteins to the profiles obtained from the plasma of 13 human SAH patients.
Following SAH, systemic IL-6 levels increased rapidly, whereas IL-10 levels were reduced. Neutrophils were increased both in the brain and in the blood reflecting local and peripheral inflammation following SAH. More intracerebral pro-inflammatory monocytes were found at early time points. Astrocyte and microglia activation were also increased, and mice had severe motor deficits, which were associated with an increase in the percentage of caspase-3-positive apoptotic neurons. Similarly, we found that IL-6 levels in patients were rapidly increased following SAH. ICAM-1, bFGF, IL-7, IL-12p40, and MCP-4 variations over time were different between SAH patients with good versus bad outcomes. Moreover, high levels of Flt-1 and VEGF at admission were associated with worse outcomes.
SAH induces an early intracerebral infiltration and peripheral activation of innate immune cells. Furthermore, microglia and astrocytic activation are present at later time points. Our human and mouse data illustrate that SAH is a systemic inflammatory disease and that immune cells represent potential therapeutic targets to help this population of patients in need of new treatments.
Besides their therapeutic benefit as cell source, neural stem/progenitor cells (NSPCs) exhibit immunosuppressive properties of great interest for modulating immune response in the central nervous ...system. To decipher the mechanisms of NSPC-mediated immunosuppression, activated T cells were exposed to NSPCs isolated from fetal rat brains. Analyses revealed that NSPCs inhibited T-cell proliferation and interferon-gamma production in a dose-dependent manner. A higher proportion of helper T cells (CD4+ T cells) was found in the presence of NSPCs, but analyses of FoxP3 population indicated that T-cell suppression was not secondary to an induction of suppressive regulatory T cells (FoxP3+ CD4+ CD25+). Conversely, induction of the high affinity interleukin-2 (IL-2) receptor (CD25) and the inability of IL-2 to rescue T-cell proliferation suggest that NSPCs display immunosuppressive activity without affecting T-cell activation. Cultures in Transwell chambers or addition of NSPC-conditioned medium to activated T cells indicated that part of the suppressive activity was not contact dependent. We therefore searched for soluble factors that mediate NSPC immunosuppression. We found that NSPCs express several immunosuppressive molecules, but the ability of these cells to inhibit T-cell proliferation was only counteracted by heme oxygenase (HO) inhibitors in association or not with nitric oxide synthase inhibitors. Taken together, our findings highlight a dynamic crosstalk between NSPCs and T lymphocytes and provide the first evidence of an implication of HO-1 in mediating the immunosuppressive effects of the NSPCs.
BackgroundMyocarditis from immune checkpoint inhibition (ICI) has been reported in 0.04–1.14% of patients on ICI, with mortality up to 50%.1 Murine models reveal cardiac-myosin-specific T cells ...contribute to ICI-myocarditis; genetic/phenotypic differences may predispose to their activity.2 We present the Montreal Immune-Related Adverse Events (MIRAE) ICI-myocarditis project, conducted by an interdisciplinary team of physicians and scientists to understand molecular drivers of ICI-myocarditis.MethodsThis case-control study comprises three groups of patients treated with ICI: 1) ICI-myocarditis; 2) non-ICI troponemia (elevated troponins from non-immune etiology); and 3) controls matched by tumour type, with no IRAEs nor troponemia. We analyzed blood samples from prior to ICI, at time of troponemia, or at 3–6 months after ICI initiation if no troponemia. We developed a multi-omics pipeline to understand mechanisms of ICI-myocarditis (figure 1), with immune cell subpopulation profiling of peripheral blood mononuclear cells (PBMCs) using single cell RNA and T/B cell receptor sequencing. This is validated with genomic DNA methylation, cytokine analyses, and PhenoCycler spatial single-cell imaging proteomics to localize cellular sources of upregulated cytokines and to visualize cell-cell interactions underpinning cardiac pathology.ResultsOf 473 patients treated with ICI in the MIRAE biobank, 3.59% had ICI-myocarditis. Of these, 19 had stored samples and were included in this study (see table 1 for baseline characteristics). 5 patients (26%) developed arrhythmias. 10 (53%) had concurrent IRAE. 1 (5%) died from concurrent IRAE. There were no deaths from myocarditis. Elevations of blood neutrophil-to-lymphocyte ratio, alanine transaminase, and aspartate aminotransferase were associated with ICI-myocarditis, compared to non-myocarditis patients at 3–6 months on ICI (figure 2). Plasma cytokine profiling of 13 ICI-myocarditis cases and matching controls demonstrated no significant differences in baseline cytokines prior to ICI. Significant elevations of chemokine IP10 and anti-inflammatory cytokine IL10 were detected at time of myocarditis, implicating various immune cells, including T lymphocytes and monocytes (figure 3). Immune cell subpopulation profiling of PBMCs is ongoing (figure 4). Spatial profiling of the first ICI-myocarditis biopsy demonstrated T cell and macrophage infiltration between myocardiocytes and granulocyte accumulation within fibrotic tissue. This suggests a role of innate immunity in myocardial damage, in addition to lymphocyte activation (figure 5).ConclusionsThis is one of the largest translational studies of ICI-myocarditis patients and matched controls. The preliminary data highlight the role of innate immunity, in addition to the previously known role of T lymphocytes. Advancing molecular understandings of ICI-myocarditis will allow us to design more targeted, effective immunosuppressive treatments for ICI-myocarditis.AcknowledgementsLaboratories of Dr Wilson Miller, Dr Sonia Del Rincón, Dr Réjean Lapointe, Dr Jun Ding, and Dr Lucas Salas.Funding from Canadian Institutes of Health Research and a generous donation to the Jewish General Hospital Clinical Research Unit by Cathy Monticciolo-Cianci in memory of her mother Maria Monticciolo.ReferencesMahmood SS, Fradley MG, Cohen JV, Nohria A, Reynolds KL, Heinzerling LM, et al. Myocarditis in Patients Treated With Immune Checkpoint Inhibitors. J Am Coll Cardiol. 2018;71(16):1755–64.Axelrod ML, Meijers WC, Screever EM, Qin J, Carroll MG, Sun X, et al. T cells specific for α-myosin drive immunotherapy-related myocarditis. Nature. 2022;611(7937):818–26.Ethics ApprovalThis study was approved by the CIUSS West-Central Montreal Ethics Board; approval number 2022–3081. All patient participants gave informed consent to be enrolled in the Montreal Immune-Related Adverse Events project.Abstract 1250 Figure 1Montreal Immune-Related Adverse Events ICI-myocarditis research pipelineAbstract 1250 Figure 2ICI-Myocarditis is associated with elevation of NLR, ALT and AST in the blood. (A-D) NLR, ALT, AST, and CK in the blood of ICI-Myocarditis cases and controls at baseline and during ICI (follow up vs. irAE). Data are shown as mean +/- standard error of the mean. Statistical analysis was performed using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001Abstract 1250 Figure 3Distinctive cytokine profile in plasma during ICI-Myocarditis. A panel of 30 cytokines, including all signature type 1, type 2 and type 3 cytokines, was used. IP-10 and IL-10 were found to be significantly elevated at time of myocarditis event, compared to controls at 3–6 month follow up on ICI. (A-E) Plasma levels of IP-10, IL-10, GMCSF, IL-15, and IL-13 in ICI-myocarditis cases versus ICI-controls. (F) Heatmap of data represented in A-E with cases organized according to grade of myocarditis. Data are shown as mean +/- SEM. Statistical analysis was performed using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001Abstract 1250 Figure 4Analysis of single-cell RNA-sequencing data from 6 patient samples. Single cell sequencing is ongoing for our 17 patients with ICI myocarditis, as well as 5 patients with troponemia without myocarditis, and 22 matched controls. The goal is to determine the composition of the PBMCs at baseline and during ICI-myocarditis, to identify potentially pathogenic cell subsets and their associated transcriptional signatures. (A) UMAP plots depicting cells from myocarditis patients, distinguished by Leiden cluster labels indicating cell subpopulations. Cells sourced from different patients were amalgamated using Harmony to mitigate potential batch effects. (B) UMAP plots demonstrating cells from myocarditis patients, categorized by the defined cell type. (C) Illustration of the biomarkers utilized for cell type annotation. (D) Representation of the cell type composition within each condition, namely, baseline, follow, and irAEAbstract 1250 Figure 5Optimized 54-marker myocarditis antibody panel for use in Highly Multiplex Phenotyping with the PhenoCycler Imaging platform. (A) List of markers. (B) Low magnification view of whole FFPE myocardial tissue section. Inset shows high magnification immunofluorescence images of MYH6 (cardiomyocytes), CD45 (immune cells), and CD68 (macrophages). (C) Fibrotic region. Inset (below) shows single channels for CD45, CD11b, CD45RO, CD15, VISTA, DAPI, and MergeAbstract 1250 Table 1Baseline characteristics
Vagal neurostimulation (VNS) is used for the treatment of epilepsy and major medical-refractory depression. VNS has neuropsychiatric functions and systemic anti-inflammatory activity. The objective ...of this study is to measure the clinical efficacy and impact of VNS modulation in depressive patients. Six patients with refractory depression were enrolled. Depression symptoms were assessed with the Montgomery-Asberg Depression Rating, and anxiety symptoms with the Hamilton Anxiety Rating Scale. Plasmas were harvested prospectively before the implantation of VNS (baseline) and up to 4 years or more after continuous therapy. Forty soluble molecules were measured in the plasma by multiplex assays. Following VNS, the reduction in the mean depression severity score was 59.9% and the response rate was 87%. Anxiety levels were also greatly reduced. IL-7, CXCL8, CCL2, CCL13, CCL17, CCL22, Flt-1 and VEGFc levels were significantly lowered, whereas bFGF levels were increased (
values ranging from 0.004 to 0.02). This exploratory study is the first to focus on the long-term efficacy of VNS and its consequences on inflammatory biomarkers. VNS may modulate inflammation via an increase in blood-brain barrier integrity and a reduction in inflammatory cell recruitment. This opens the door to new pathways involved in the treatment of refractory depression.
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