Immune Cell Subpopulations Research Articles

Single-cell RNA-seq landscape midbrain cell responses to red spotted grouper nervous necrosis virus infection.

Viral nervous necrosis (VNN) is an acute and serious fish disease caused by nervous necrosis virus (NNV) which has been reported massive mortality in more than fifty teleost species worldwide. VNN causes damage of necrosis and vacuolation to central nervous system (CNS) cells in fish. It is difficult to identify the specific type of cell targeted by NNV, and to decipher the host immune response because of the functional diversity and highly complex anatomical and cellular composition of the CNS. In this study, we found that the red spotted grouper NNV (RGNNV) mainly attacked the midbrain of orange-spotted grouper (Epinephelus coioides). We conducted single-cell RNA-seq analysis of the midbrain of healthy and RGNNV-infected fish and identified 35 transcriptionally distinct cell subtypes, including 28 neuronal and 7 non-neuronal cell types. An evaluation of the subpopulations of immune cells revealed that macrophages were enriched in RGNNV-infected fish, and the transcriptional profiles of macrophages indicated an acute cytokine and inflammatory response. Unsupervised pseudotime analysis of immune cells showed that microglia transformed into M1-type activated macrophages to produce cytokines to reduce the damage to nerve tissue caused by the virus. We also found that RGNNV targeted neuronal cell types was GLU1 and GLU3, and we found that the key genes and pathways by which causes cell cytoplasmic vacuoles and autophagy significant enrichment, this may be the major route viruses cause cell death. These data provided a comprehensive transcriptional perspective of the grouper midbrain and the basis for further research on how viruses infect the teleost CNS.

Immune Profile of Sickle Cell Anemia Patients Treated with Allogeneic Stem Cell Transplantation

Sickle cell disease (SCD) is one the most common recessive monogenic disease in the world. In the last years, SCD patients have been successfully treated with allogeneic hematopoietic stem cell transplantation (HSCT), the only curative option to date. However, the immune reconstitution mechanisms involved in therapeutic response of HSCT in SCA patients are not elucidated yet. For that, blood samples from SCD patients (N=24) were collected before (pre-tx) and until 1 year after transplantation (1 month, 3, 6 and 12 months) and immune cell subpopulations were analyzed by flow cytometry. In addition, peripheral blood mononuclear cells from 4 SCD patients and 4 healthy individuals were isolated, RNA was isolated and cDNA synthetized, and expression of genes associated with inflammatory response was evaluated by PCR array. Serum cytokines/chemokine levels were also measured in SCD patients by luminex assays. We observed decreased number of total lymphocytes until 3 months after HSCT (mean±SD: 1496±1012 cells/uL) compared to pre-tx (2671±924.8; p<0.05), as well as B lymphocytes (pre-tx: 305.7±239; 3 months: 132.2±193.7; p<0.05). The T lymphocytes counts decreased until 1 month after transplantation (pre-tx: 788.3±537.3; 1 month: 327.9±461; p<0.05) and CD4 T cells numbers reduced until 6 months (pre-tx: 487±337.3; 6 months: 281.3±222.7; p<0.05), while CD8 T cells counts did not change, resulting in significant inversion of CD4/CD8 ratio until 1 year after transplantation (1.11±0.58) compared to Pre-tx (2.05±0.77; p<0.05). Specifically, naïve CD4+ T cell number decreased until 6 months after transplantation (pre-tx: 182.6±146.9; 6 months: 91.33±90,42; p<0.05) and central memory CD4+ T cell until 3 months (pre-tx: 224.7±160; 3 months: 77.42±75.74; p<0.05). Interestingly, the numbers of RTE was lower until 3 months after HSCT (55.43±59.88) than pre-tx (136±108.7; p<0.05), but baseline value was achieved after this period, suggesting the generation of new naïve T cells by thymus after transplantation. Effector CD4+ T cell increased 6 months after HSCT (pre-tx: 20.03±18.67; 6 months: 39.89±31.24; p<0.05) and effector CD8+ T cell 12 months (pre-tx: 64.83±64.97; 12 months: 174.7±193.2; p<0.05). In addition, the reconstitution of regulatory T cell subsets was also studied. The absolute number of CD4+CD25+CTLA-4+ T cell (pre-tx: 118.5±95.16; 6 months: 54.37±42.25) and CD4+CD25+Foxp3+ T cell (pre-tx: 112.4±78.96; 6 months: 56.55±46.84) decreased until 6 months after transplantation compared to pre-tx (p<0.05), but reached baseline counts 1 year after HSTC. The number of CD4+CD25+GITR+ T cell and CD8+CD28-Foxp3+ T cell did not change after transplant. However, increased number of CD8+CD28- T cells was observed 6 (307.6±382.6) and 12 months (373.3±357.5) after transplantation compared to pre-tx (120.8±133.8) and CD8+CD28-CD57+ T cells raised 12 months (pre-tx: 52.69±92.86; 12 months: 160.5±175.9; p<0.05). Clustering analysis demonstrated that gene expression profiling of SCD patients at 1 year after transplantation was similar to healthy individuals. Expressions of chemokine genes (such as CCL2, CCL7, CXCL1, CXCL2, CXCL3, CXCL8) and/or chemokine receptors (MIP-1alpha, MCP-3, CCL5, IL-8) important for leukocytes recruitment were increased in SCD patients before transplant compared to healthy subjects. Indeed, the serum concentration of MIP-1α (control: 3.64±1.07pg/mL; pre-tx: 6.4±2.54) and IL-8 (control: 0±0; pre-tx: 7.4±8.69) was also higher in patients before transplantation compared to controls (p<0.05). However, one month after transplantation the expression of chemokine genes CXCL1, CXCL2, CXCL3, CXCL8, CCL2 and CCL7 was decreased when compared to pre-transplantation levels. One year after transplantation, the expression of chemokine genes (CCL4, CCL5 and CCL16) and proinflammatory cytokines (IL-6, TNF and IFN-γ) increased compared to pre-tx, however the gene expression of chemokine CCL7, CXCL1, CXCL2, CXCL3, CXCL8 remained decreased. In addition, serum levels of MIP-1α decreased until 12 months after transplantation (3.82±3.34) compared to pre-transplant (6.4±2.54). Thus, our data suggest that HSCT induces alterations in immune and inflammatory profile of SCD patients after HSCT, such as downregulation of the chemokine genes expression and increase of regulatory T cell subsets, besides the correction of the genetic disease. DisclosuresHermine:AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; Hybrigenics: Research Funding; Novartis: Research Funding; INatherys: Equity Ownership, Research Funding; Celgene: Research Funding.

Defining a Bone Marrow Immune Signature Associated with Immunoglobulin Isotype Switching after Autologous Stem Cell Transplant

Triplet induction therapy followed by autologous stem cell transplant (ASCT) and maintenance therapy can render long-term remission for newly diagnosed multiple myeloma (MM) patients (pts). Robust post-ASCT immune-reconstitution correlates with lower minimal residual disease (MRD) and better clinical outcomes but immune signatures to define such responses are lacking (Ho et al. Blood Advances 1.15 (2017). Following ASCT, the Immunoglobulin Isotype Switch (IIS) results in a new serum M-protein on serum protein electrophoresis (SPE), distinct from the M-protein pattern present at diagnosis, may be associated with a good prognosis. The objective of this study was to analyze humoral immune reconstitution that presents as IIS after ASCT and to determine if there was a correlation with patient or disease characteristics, transplant parameters and to define the associated immune signature.MethodsThe Institutional Review Board-approved study evaluated MM pts who underwent ASCT between 2007 and 2016. Data collection included patients' characteristics, induction therapy, mobilization regimen, graft characteristics including CD34 cell dose, myeloid and erythroid progenitor cells (multi-potential colony forming unit or CFU-GM and erythroid burst-forming unit (BFU-E). Percentage of NK cells (CD56(+) CD3(-) and CD16(+)CD3(-)), T-cells (CD3(+),CD56 (-)), B-cells (CD19(+)),and T-cell subpopulations (CD4(+), CD8(+),were measured with flow cytometry in pre- and post-ASCT bone marrow samples. IIS was defined as the appearance of a new serum monoclonal spike on SPE and immunofixation different from original heavy or light chain detected at diagnosis. Monoclonal proteins and quantitative immunoglobulins were assessed day+100 and with frequency of 2-8 times per year afterward.The start date for all time dependent variables was the day of transplant. The Kaplan-Meier method was used to assess overall survival (OS) and progression-free survival (PFS). Two-tailed log-rank tests compared OS and PFS curves. Factors with a P -value <0.2 in univariate analysis were included in multivariate analysis, which was performed using the Cox regression hazard model. All analyses were performed utilizing SAS 9.2 (SAS Institute, Cary, NC, USA).ResultsThe analysis included 177 patients with median age of 60.7 years. IIS was detected in 39 pts (22.0%) with a significantly higher incidence in pts who received lenalidomide before ASCT (11.2% vs. 30.4%, P=0.001). Post-ASCT IIS was more frequent in IgA, light chain disease, and in pts who experience very good partial and complete response post-ASCT rather than less optimal response (Table-1 and 2). The occurrence of IIS did not correlate with ASCT toxicity, i.e., mucositis duration or grade as well as length of hospital admission (Table 2). IIS occurred more frequently in pts without suppressed un-involved immunoglobulin (92% vs 8%, p=0.001).The presence of post-ASCT IIS correlated significantly with PFS and OS (Figure.1.A and B). Age, cytogenetics, response category, presence of IIS and low LDH correlated with PFS by univariate analysis. Cytogenetics, LDH and IIS presence were also significantly associated with response as determined by multivariate analysis. The median time between pre- and post-ASCT BM was 92 days (range 79-123). Pts with IIS showed a trend toward faster B cell recovery in comparison to pts without IIS, however this difference did not reach statistical significance. All pts had low CD4/CD8 ratio, due to low number of CD4 and higher number of CD8. Appearance of post-ASCT IIS was associated with statistically lower CD8 T cell percentages and higher CD4/CD8 ratio in comparison to pts without IIS (Figure.1.C), suggestive of faster immune reconstitution in this group of pts. All pts who relapsed had an isotype different from IIS, highlighting the benign nature of this phenomenon.ConclusionOur data are consistent with the benign nature of IIS that may emerge in certain patients following ASCT. Association of a robust recovery of specific immune cell subpopulations with IIS occurrence and superior survival outcomes may represent an anti-myeloma protective effect resulted from cellular and humoral immunoreconstitution. Efforts to better understand how specific immune subpopulations trigger and regulate the immune response to tumors may improve long-term control of MM. Further research to define underlying anti-tumor effect of ISS is needed. [Display omitted] DisclosuresDe Lima:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Research Funding. Malek:Sanofi: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Lateral dimension and amino-functionalization on the balance to assess the single-cell toxicity of graphene on fifteen immune cell types

Given the wide variety of potential applications of graphene oxide (GO), its consequent release into the environment poses serious concerns on its safety. The future production and exploitation of graphene in the years to come should be guided by its specific chemical-physical characteristics. The unparalleled potential of single-cell mass cytometry (CyTOF) to dissect by high-dimensionality the specific immunological effects of nanomaterials, represents a turning point in nanotoxicology. It helps us to identify the safe graphene in terms of physical-chemical properties and therefore to direct its future safe production.Here we present a high-dimensional study to evaluate two historically indicated as key parameters for the safe exploitation: functionalization and dimension. The role of lateral dimension and the amino-functionalization of GO on their immune impact were here evaluated as synergistic players. To this end, we dissected the effects of GO, characterized by a large or small lateral size (GO 1.32 μm and GO 0.13 μm, respectively), and its amino-functionalized counterpart (GONH2 1.32 μm and GONH2 0.13 μm, respectively) on fifteen cell types of human primary peripheral blood mononuclear cells (PBMCs).We describe how the smallest later size not only evokes pronounced toxicity on the pool of PBMCs compared to larger GOs but also towards the distinct immune cell subpopulations, in particular on non-classical monocytes, plasmacytoid dendritic cells (pDCs), natural killer cells (NKs) and B cells. The amino-functionalization was able to improve the biocompatibility of classical and non-classical monocytes, pDCs, NKs, and B cells. Detailed single-cell analysis further revealed a complex interaction of all GOs with the immune cells, and in particular monocyte subpopulations, with different potency depending on their physicochemical properties. Overall, by high-dimensional profiling, our study demonstrates that the lateral dimension is an important factor modulating immune cells and specifically monocyte activation, but a proper surface functionalization is the dominant characteristic in its immune effects. In particular, the amino-functionalization can critically modify graphene impact dampening the immune cell activation. Our study can serve as a guide for the future broad production and use of graphene in our everyday life.

Tumor Microenvironment Characteristics of Pancreatic Cancer to Determine Prognosis and Immune-Related Gene Signatures.

Background: Pancreatic cancer (PC) is one of the most lethal types of cancer with extremely poor diagnosis and prognosis, and the tumor microenvironment plays a pivotal role during PC progression. Poor prognosis is closely associated with the unsatisfactory results of currently available treatments, which are largely due to the unique pancreatic tumor microenvironment (TME). Methods: In this study, a total of 177 patients with PC from The Cancer Genome Atlas (TCGA) cohort and 65 patients with PC from the GSE62452 cohort in Gene Expression Omnibus (GEO) were included. Based on the proportions of 22 types of infiltrated immune cell subpopulations calculated by cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT), the TME was classified by K-means clustering and differentially expressed genes (DEGs) were determined. A combination of the elbow method and the gap statistic was used to explore the likely number of distinct clusters in the data. The ConsensusClusterPlus package was utilized to identify radiomics clusters, and the samples were divided into two subtypes. Result: Survival analysis showed that the patients with TMEscore-high phenotype had better prognosis. In addition, the TMEscore-high had better inhibitory effect on the immune checkpoint. A total of 10 miRNAs, 311 DEGs, and 68 methylation sites related to survival were obtained, which could be biomarkers to evaluate the prognosis of patients with pancreatic cancer. Conclusions: Therefore, a comprehensive description of TME characteristics of pancreatic cancer can help explain the response of pancreatic cancer to immunotherapy and provide a new strategy for cancer treatment.

Neoplastic and immune single-cell transcriptomics define subgroup-specific intra-tumoral heterogeneity of childhood medulloblastoma.

Medulloblastoma (MB) is a heterogeneous disease in which neoplastic cells and associated immune cells contribute to disease progression. We aimed to determine the influence of neoplastic and immune cell diversity on MB biology in patient samples and animal models. To better characterize cellular heterogeneity in MB we used single-cell RNA sequencing, immunohistochemistry, and deconvolution of transcriptomic data to profile neoplastic and immune populations in patient samples and animal models across childhood MB subgroups. Neoplastic cells cluster primarily according to individual sample of origin which is influenced by chromosomal copy number variance. Harmony alignment reveals novel MB subgroup/subtype-associated subpopulations that recapitulate neurodevelopmental processes, including photoreceptor and glutamatergic neuron-like cells in molecular subgroups GP3 and GP4, and a specific nodule-associated neuronally differentiated subpopulation in the sonic hedgehog subgroup. We definitively chart the spectrum of MB immune cell infiltrates, which include subpopulations that recapitulate developmentally related neuron-pruning and antigen-presenting myeloid cells. MB cellular diversity matching human samples is mirrored in subgroup-specific mouse models of MB. These findings provide a clearer understanding of the diverse neoplastic and immune cell subpopulations that constitute the MB microenvironment.

MO074TILVESTAMAB, A FUNCTION-BLOCKING MONOCLONAL ANTIBODY INHIBITOR OF AXL RTK SIGNALLING, LIMITS THE ONSET OF RENAL FIBROTIC CHANGES IN HUMAN KIDNEYS EX VIVO

Abstract Background and Aims Interstitial fibrosis, characterised by the accumulation of extracellular matrix in the cortical interstitium, is directly correlated with progressive chronic kidney disease secondary to inflammatory, immunologic, obstructive or metabolic causes. An invariant histologic marker of this progression is the accumulation of fibroblasts, with the phenotypic appearance of activated myofibroblasts expressing alpha smooth muscle actin (αSMA) within intracellular contractile stress fibres. Once present, these myofibroblasts are prognostic indicators of expansion of fibrotic matrix and progressive tubular atrophy, leading towards end-stage disease. The Receptor Tyrosine Kinase AXL is involved in a range of kidney pathologies, with increased activity associated with Epithelial to Mesenchymal Transition (EMT) and tubular proliferation following podocyte loss. In mice treated with an angiotensin-converting enzyme (ACE) inhibitor, enhancement of AXL expression is localised to tubular segments within the medulla and there is evidence of parallel regulatory control of ACE and AXL. We have demonstrated enhanced expression of AXL and the mesenchymal marker, vimentin in diseased human kidney tissue secondary to diabetes or hypertension. Targeting AXL with a small-molecule inhibitor has previously been reported to attenuate fibrosis and reduce inflammation in the unilateral ureteric-outflow obstruction (UUO) model of kidney fibrosis in mice (Landolt et al., 2019). Tilvestamab is a novel function blocking humanized anti-AXL antibody. Tilvestamab blocks GAS6-mediated AXL receptor activation in fibroblasts and renal tubule epithelial cells and mediates AXL receptor internalization and degradation. In this study we aimed to further characterise AXL as a target in CKD and to investigate anti-fibrotic efficacy of tilvestamab. Method Eight weeks old male C57BL/6 mice underwent UUO operation. After 15 days, kidneys were dissociated and stained with a high dimensional single cell mass cytometry 33 markers antibody panel. Data were analysed using JMP Genomics (v.8.2). Precision Cut Kidney Slices (PCKSs) from explanted human kidney tissue were propagated in a bioreactor (Paish et al., 2019, FibroFind, UK). PCKS were incubated for 72hrs in the presence of investigational drugs. Secreted collagen1a1 were quantified by ELISA. RNA was reverse transcribed to cDNA and used in qPCRs to measure Col1a1 and αSMA. FFPE sections were stained for αSMA. High magnification images were taken of each slide and analysed for surface area covered by the stain. Results Expression pattern of AXL during development of kidney fibrosis in the UUO model was investigated using a mass cytometry antibody panel designed for identifying subpopulations of immune cells as well as cell populations of the fibrotic stroma. Two predominant cell populations were affected by ligation; the mesenchymal and the immune island. AXL was a marker characterising several of the key populations that expanded upon ligation supporting a role for AXL in kidney fibrosis pathogenesis. In an ex vivo model of human PCKS, tilvestamab dose-dependently reduced the levels of αSMA. When combined with the lower of two doses of the ACE inhibitor enalapril, the lowest dose of tilvestamab synergized to reduce αSMA levels further as well as reducing secreted Collagen 1a1. Conclusion AXL expression is induced in key cell populations during development of kidney fibrosis supporting AXL as a novel target in CKD. Tilvestamab represents a promising strategy for the pharmacologic intervention of kidney fibrosis, and the potential synergy with current reno-protective therapies warrants further exploration.

MO695THE ASSOCIATION OF CIRCULATING CD14++CD16+ MONOCYTES, NATURAL KILLER CELLS AND REGULATORY T CELLS SUBPOPULATIONS WITH CARDIOVASCULAR DISEASE IN A COHORT OF PERITONEAL DIALYSIS PATIENTS

Abstract Background and Aims Advanced chronic kidney disease (CKD) is characterized by elevated expression of the proinflammatory and pro-atherogenic CD14++CD16+ monocytes subset. The role of lymphocyte subpopulations including natural killer (NK) cells and CD4+CD25+ regulatory T cells (Tregs) in the modulation of inflammation and immunity and subsequent cardiovascular implications have received increasing attention. The role of immune cell subpopulations remains to be determined in peritoneal dialysis (PD) patients. The aim of this pilot study was to investigate potential correlations between blood levels of CD14++CD16+ monocytes, NK cells and Tregs with phenotypes of established cardiovascular disease (CVD), including coronary artery disease (CAD) and heart failure (HF) in a cohort of PD patients. Method 29 stable PD patients (mean age 66.96 years ±14.5, 62% males) were enrolled. Exclusion criteria were a history of malignancy, autoimmune disease, active or chronic infections and a recent (&amp;lt; 3 months) cardiovascular event. Demographic, laboratory and bioimpedance measurements data (overhydration, extracellular and total body water and their ratios) were collected. The analysis of peripheral blood immune cell subsets was performed using flow cytometry (FC). Additionally, in 7 PD patients the distribution of the immune cells was evaluated by FC at two time points: T0 (before initiation of PD - CKD stage G5) and T1 (after PD start). Results The median dialysis vintage was 34.5 (range 3.2-141) months. Overall, PD patients had 527 ± 199 monocytes and 1731 ± 489 lymphocytes while mean percentage of CD14++CD16+ monocytes was 9.3 ±6.36% (normal range 2-8%), NK cells 16.6±10.3% (normal range 5-15%) and Tregs 2.1±1.76% (normal range 1-3%). There was no correlation of either of these cell subpopulations with age, PD vintage, inflammation markers (CRP, fibrinogen, albumin, hsTroponin-I), overhydration markers or comorbidities. Only increased NK cells were associated with the presence of HF in PD (24.87 vs 14.92%, p 0.047). In multiple regression analysis, NK cells levels were strongly associated with the presence of edema (beta coef=13.7, p&amp;lt;0.001) and CAD (beta coef=7.1, p=0.046). At T0 mean percentage of CD14++CD16+ monocytes, NK cells and Tregs were 9.7 ±4.5%, 17.1 ±3.84% and 2.38± 1.26% respectively whereas at T1 mean percentage of CD14++CD16+ monocytes was 13.3% ±8.4, NK cells 19.8±6.47% and Tregs 1.5±0.6%. Paired t-test of cell subpopulations (T0 vs T1) showed that only the Tregs were significantly decreased (p =0.018), while the other subpopulations did not differ and remained increased. Conclusion Our study is the first to evaluate the potential association between specific immune cell subsets and cardiovascular disease in long-term PD patients. Increased NK cells levels directly correlate both with the presence of HF and CAD in PD patients. Longitudinal results suggest that CD14++CD16+ and NK cells remain increased after PD start, while Tregs decrease further. The state of pro-inflammation and immune deregulation appear to persist after initiating PD. Future research is required to evaluate the role of immune cells subsets as potential tools to identify patients who are at the highest risk for complications and to guide interventions that may improve clinical outcomes.

Identification of a predictive biomarker and two pharmacodynamic biomarkers to ADG106 treatment, a novel anti-CD137 agonist antibody, in phase I clinical trials.

e14505 Background: ADG106 is a fully human agonistic anti-CD137 monoclonal IgG4 antibody that mediates anti-tumor activities via unique mechanisms of action. Here we provide safety and efficacy updates from our phase I trials and report the findings of a predictive biomarker and two pharmacodynamic biomarkers which correlate with patients’ clinical responses to ADG106 treatment and demonstrate target engagement, respectively. Methods: Formalin fixed and paraffin embedded (FFPE), blood and plasma specimens were collected from 92 patients enrolled in our phase I trials. We measured expression across a panel of protein biomarkers in FFPE specimens using three highly sensitive detection technologies: multiple immunohistochemical (IHC) staining of protein expression, the BD Multitest 6-color TBNK reagent for profiling immune cell subpopulations, and the MSD-ECL electrochemiluminescence assay for detection of soluble CD137. Objective tumor responses were determined using RECIST v1.1 for solid tumor patients and Lugano classification for lymphoma patients. Results: As of November 30, 2020, ADG106 has demonstrated a favorable safety profile and efficacy in the phase I clinical trials with a disease control rate of 56%. From a retrospective analysis of 28 pretreatment FFPE specimens, we identified a predictive biomarker that correlated with tumor shrinkage upon ADG106 treatment. We identified four biomarker positive specimens from two patients with lymphoma and two with solid tumors. Three out of four biomarker positive patients achieved greater than 30% tumor shrinkage after 3mg/kg or 5mg/kg ADG106 treatment. One biomarker positive patient with stable disease received a low dose ADG106 treatment at 0.5mg/kg during dose escalation. None of the 24 biomarker negative patients showed significant clinical response. A tissue microarray study confirmed expression of this predictive biomarker in a variety of tumor types suggesting a broad indication for ADG106 therapy. Our biomarker studies also demonstrated target engagement with increased NK cell proliferation and soluble CD137 upon ADG106 treatment. Analysis of safety, efficacy, PK and PD data allowed us to select a recommended dose for the upcoming phase II study. Conclusions: We identified a biomarker predictive of response to antitumor CD137 blockade by ADG106, as well as demonstrated the involvement of NK cells in ADG106 mediated anti-tumor activities. In upcoming phase II trials, we plan to enrich for populations expressing this predictive biomarker to demonstrate a clinical benefit to ADG106 therapy further validating early biomarker-based patient stratification. We will also explore the potential of selecting patients for combination treatment with anti-PD-1 therapies. Clinical trial information: NCT03802955.

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications.

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CAR-T cell manufacturing process.

Single-cell RNA Sequencing Reveals Heterogeneity Amongst Immune Cell Subpopulations in LPS-Induced ALI Mice treated with I3C

Abstract Traditionally, macrophage M1-vs-M2 dichotomy have remained fundamentally unchanged since their identification. The oversimplification of immune cell subpopulations using flow cytometric analysis fails to capture the full heterogeneity of these cells in response to disease state and treatment. However, single-cell RNA sequencing (sc-RNAseq) serves as a diagnostic tool that clusters cells based on the whole transcriptomics of individual cells, without the limitation of surface markers and transcriptional factors. In the current study, we aimed to better understand the immunomodulatory mechanism of indole-3-carbinol (I3C) on cell state. To that end, 5mg/kg of LPS was intranasally injected in C57BL/6 mice to induce ALI, and the mice were treated with 80mg/kg I3C three hours after disease induction. After 72 hours mice were euthanized. Whole lung cells were utilized for flow cytometry and sc-RNAseq. Strikingly, the traditional M1-vs-M2 separation was inadequate in classifying all macrophage clusters. LPS+I3C treated-mice had a decrease in Isg15, Ccl5, and Ly6c2 expression in M1 cluster; genes linked to inflammation. Also, the expression of monocyte chemoattractant genes (Cxcl2 and Cxcl3), arginine-associated transport gene (Slc7ac), and major histocompatibility complex (MHC) genes (H2-Ab1 and H2-K1) in M2 cluster was decreased in the diseased group following I3C treatment. In conclusion, several alterations in gene expression in myeloid immune cell subpopulations were identified following treatment with indole-3-carbinol. Thus, shedding light on several new macrophage subpopulations not identified using traditional flow cytometric analysis may offer novel mechanistic insights into disease induction.

Immune Phenotyping of Patients With Acute Vogt-Koyanagi-Harada Syndrome Before and After Glucocorticoids Therapy.

Previous studies have established that disturbed lymphocytes are involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) syndrome. Accordingly, glucocorticoids (GCs), with their well-recognized immune-suppressive function, have been widely used for treatment of VKH patients with acute relapses. However, the systemic response of diverse immune cells to GC therapy in VKH is poorly characterized. To address this issue, we analyzed immune cell subpopulations and their phenotype, as well as cytokine profiles in peripheral blood from VKH patients (n=25) and health controls (HCs, n=21) by flow cytometry and luminex technique, respectively. For 16 patients underwent GC therapy (methylprednisolone, MP), the aforementioned measurements as well as the transcriptome data from patients before and after one-week’s GC therapy were also compared to interrogate the systemic immune response to GC therapy. Lymphocyte composition in the blood was different in VKH patients and HCs. VKH patients had significantly higher numbers of T cells with more activated, polarized and differentiated phenotype, more unswitched memory B cells and monocytes, as compared to HCs. MP treatment resulted in decreased frequencies of T cells and NK cells, inhibited NK cell activation and T cell differentiation, and more profoundly, a marked shift in the distribution of monocyte subsets. Collectively, our findings suggest that advanced activation and differentiation, as well as dysregulated numbers of peripheral lymphocytes are the major immunological features of VKH, and GC therapy with MP not only inhibits T cell activation directly, but also affects monocyte subsets, which might combinatorically result in the inhibition of the pathogenic immune response.

Patterns of immune infiltration in gastric cancer and their clinical significance.

The malignant phenotypes of cancer are defined not only by its intrinsic tumor cells but also by the tumor-infiltrating immune cells activated and recruited to the cancer microenvironment. However, a comprehensive introduction of gastric cancer immune cell infiltration has not been identified so far. In this study, we comprehensively analyzed the tumor-infiltrating immune cells abundance in gastric cancer for the first time by CIBERSORT. The meta-analysis, single-sample gene set enrichment analysis and hierarchical agglomerative clustering were used to measure and evaluate the respective proportions of 22 cell types of immune infiltration using normalized gene expression data. The fraction of tumor-infiltrating immune cells subpopulations was also evaluated to determine the associations with clinical features and molecular subtypes. Tumor-infiltrating immune cells are extensively involved in the pathogenesis and development of the gastric cancer. We discovered Tfh and activated CD4+ memory T cells were associated with poorer overall survival and Progression-free survival (PFS), but that naïve B cells were opposite for PFS. Unsupervised clustering analysis revealed there existed three tumor-infiltrating immune cells subgroups with distinct survival patterns. Specially, cluster 1 showed significantly better clinical outcome than other two clusters. Collectively, our data explored the differences of tumor-infiltrating immune cells in gastric cancer, and these variations were likely to be important clues for prognosis and management of its future clinical implementation.

Standardized Uptake Value for 18F-Fluorodeoxyglucose Is a Marker of Inflammatory State and Immune Infiltrate in Cervical Cancer.

Chemoradiotherapy for locally advanced cervical cancer fails in over a third of patients. Biomarkers with therapeutic implications are therefore needed. We investigated the relationship between an established prognostic marker, maximum standardized uptake value (SUVmax) on 18F-fluorodeoxyglucose positron emission tomography, and the inflammatory and immune state of cervical cancers. An SUVmax most prognostic for freedom from progression (FFP) was identified and compared with known prognostic clinical variables in a cohort of 318 patients treated with definitive radiation with prospectively collected clinical data. Gene set enrichment analysis (GSEA) and CIBERSORT of whole-transcriptome data from 68 patients were used to identify biological pathways and immune cell subpopulations associated with high SUVmax. IHC using a tissue microarray (TMA, N = 82) was used to validate the CIBERSORT findings. The impact of macrophages on cervical cancer glucose metabolism was investigated in coculture experiments. SUVmax <11.4 was most prognostic for FFP (P = 0.001). The GSEA showed that high SUVmax is associated with increased gene expression of inflammatory pathways, including JAK/STAT3 signaling. CIBERSORT and CD68 staining of the TMA showed high SUVmax tumors are characterized by a monocyte-predominant immune infiltrate. Coculture of cervical cancer cells with macrophages or macrophage-conditioned media altered glucose uptake, and IL6 and JAK/STAT3 signaling contribute to this effect. SUVmax is a prognostic marker in cervical cancer that is associated with activation of inflammatory pathways and tumor infiltration of myeloid-derived immune cells, particularly macrophages. Macrophages contribute to changes in cervical cancer glucose metabolism.See related commentary by Williamson et al., p. 4136.

Brain-Derived Neurotrophic Factor and Immune Cells in Osteoarthritis, Chronic Low Back Pain, and Chronic Widespread Pain Patients: Association with Anxiety and Depression.

Background and Objectives: Musculoskeletal dysfunction can induce several types of chronic pain syndromes. It is of particular interest to elucidate the pathomechanism of different forms of chronic pain. It is possible that patients who have developed chronic widespread pain (CWP) may endure different pathomechanisms as compared to those who suffer from local pain (osteoarthritis, OA) and regional pain (chronic low back pain, cLBP), especially with regard to pain regulation and its related biomediators. The aim of this study was to determine the differences in pathomechanisms among these patients by measuring pain-related biomediators, particularly brain-derived neurotrophic factor (BDNF). Additionally, subpopulations of immune cells were determined in parallel. Materials and Methods: Patients and healthy subjects (HSs) were recruited (age and gender-matched). BDNF was measured from serum samples of patients and HSs and the data of body composition parameters were recorded. Additionally, both patients and HSs were asked to fill in questionnaires related to pain intensity, anxiety, and depression. Results: Our results highlight that the levels of both free and total BDNF are significantly lower in pain patients compared to HSs, with p values of 0.041 and 0.024, respectively. The number of CD3− CD56bright natural killer (NK) cells shows significant differences between the groups. Comparing all chronic pain patients with HSs reveals a significantly lower number of CD4+ CD8+ T cells (p = 0.031), CD3− CD56bright NK cells (p = 0.049) and CD20+ CD3− cells (p = 0.007). Conclusions: To conclude, it seems that a general conformity between the pathomechanisms of different chronic pain diseases exists, although there are unique findings only in specific chronic pain patients.

Profiles of Immune Cell Infiltration in Carotid Artery Atherosclerosis Based on Gene Expression Data.

Since immune infiltration is closely associated with the progression and prognosis of atherosclerosis, we aimed to describe the abundance of 24 immune cell types within atherosclerotic tissues. In the current study, we used the Immune Cell Abundance Identifier (ImmuCellAI), a web-based tool, to estimate the abundance of 24 immune cells based on the microarray profiles of atherosclerotic carotid artery samples to analyze the proportions and the dysregulation of immune cell types within carotid atherosclerosis. We found that atherosclerotic immune cells had a diverse landscape dominated by T cells and myeloid cells and that macrophages and dendritic cells (DCs) showed different abundance in normal and atherosclerotic tissues. Moreover, the expression of macrophages was closely related to the level of the expression of DCs and of exhausted T cells, while the expression of T-helper type 1 (Th1) cells was strongly correlated with the expression of T-helper type 2 (Th2) cells and effector memory cells. Our data confirm a distinct profile of atherosclerosis-infiltrating immune cell subpopulations, which may inspire an immunological direction for research on atherosclerosis.

A flow cytometric approach to study glucocorticoid receptor expression in immune cell subpopulations of genetically engineered mice

Glucocorticoids (GCs) constitute one of the most powerful classes of anti-inflammatory agents and are used for the treatment of a plethora of diseases related to autoimmunity, allergy, cancer, and infection. In the last two decades, multiple studies using genetically engineered mice with targeted deletions of the GC receptor (GR) in individual cell types have provided insights into the mechanisms of GCs in the control of the immune system. The characterization of GR expression in these mouse models, however, mostly relied on the analysis of mRNA expression or reporter gene activity. In contrast, approaches directly detecting the GR protein on a cellular level are scarce. Thus, we here used a flow cytometric method to analyze mice in which the GR gene locus was disrupted with the help of a Cre recombinase expressed under the control of either the lck or the lysM promoter. Measuring GR protein expression in immune cell subpopulations unveiled an efficient and highly selective depletion in both strains of knock-out mice in accordance with the expected cellular specificity of the employed promoters for T cells or myeloid cells, respectively. The flow cytometric data were well in line with those from the analysis of GR mRNA expression in magnetically sorted immune cell subpopulations but they could be obtained much more quickly. In summary, our data indicate that flow cytometry is a powerful tool with which to define GR protein content at a single cell level when studying the function of GCs in the immune system.

Resident immune cells of the avascular lens: Mediators of the injury and fibrotic response of the lens.

Tissues typically harbor subpopulations of resident immune cells that function as rapid responders to injury and whose activation leads to induction of an adaptive immune response, playing important roles in repair and protection. Since the lens is an avascular tissue, it was presumed that it was absent of resident immune cells. Our studies now show that resident immune cells are a shared feature of the human, mouse, and chicken lens epithelium. These resident immune cells function as immediate responders to injury and rapidly populate the wound edge following mock cataract surgery to function as leader cells. Many of these resident immune cells also express MHCII providing them with antigen presenting ability to engage an adaptive immune response. We provide evidence that during development immune cells migrate on the ciliary zonules and localize among the equatorial epithelial cells of the lens adjacent to where the ciliary zonules associate with the lens capsule. These findings suggest that the vasculature‐rich ciliary body is a source of lens resident immune cells. We identified a major role for these cells as rapid responders to wounding, quickly populating each wound were they can function as leaders of lens tissue repair. Our findings also show that lens resident immune cells are progenitors of myofibroblasts, which characteristically appear in response to lens cataract surgery injury, and therefore, are likely agents of lens pathologies to impair vision like fibrosis.

Comprehensive analysis of autophagy-related genes and patterns of immune cell infiltration in valvular atrial fibrillation

BackgroundThe development of atrial fibrillation (AF) following valvular heart disease (VHD) remains a common disease and is associated with substantial adverse complications. However, valid molecular diagnostic and therapeutic tools for post-VHD AF have not been fully established. This study was conducted to discover the molecular mechanisms and immune microenvironment underlying AF following VHD.MethodsGene expression profiles of the GSE41177 dataset were assessed to construct a protein–protein interaction network, and then, autophagy-related hub genes were identified. In addition, to determine the functions of immune cell infiltration in valvular AF, we used the CIBERSORT algorithm to estimate the composition of 22 immune cell types in valvular heart disease. Finally, correlation analysis was carried out to identify the relationship between differentially expressed autophagy-related genes (DEARGs) and significant immune cell subpopulations to reveal potential regulatory pathways.ResultsA total of 153 DEARGs were identified in AF-VHD patients compared with controlled donors. Moreover, we screened the top ten hub nodes with the highest degrees through a network analysis. The ten hub nodes were considered hub genes related to AF genesis and progression. Then, we revealed six significant immune cell subpopulations through the CIBERSORT algorithm. Finally, correlation analysis was performed, and six DEARGs (BECN1, GAPDH, ATG7, MAPK3, BCL2L1, and MYC) and three immune cell subpopulations (T cells CD4 memory resting, T cells follicular helper, and neutrophils) were identified as the most significant potential regulators.ConclusionThe DEARGs and immune cells identified in our study may be critical in AF development following VHD and provide potential predictive markers and therapeutic targets for determining a treatment strategy for AF patients.