Cell Signature Research Articles

The immunomodulatory impacts of endocrine therapy on host immunity in early-stage hormone receptor positive, HER2 negative breast cancer.

572 Background: Endocrine therapy (ET) reduces mortality in hormonally driven breast cancer (BC). The impacts of ET on the estrogen receptor are well known; however, there is limited data on the impact of ET on the host's immune system. This project aims to elucidate the potential immunomodulatory effects of ET-induced estrogen deprivation in HR+/HER2 negative early-stage breast cancer patients (pts) as well as baseline differences between BC pts and healthy controls (HC). Methods: Pts with Stage I-III HR+/HER2- BC initiating ET after completion of local regional therapy and/or chemotherapy were enrolled (NCT03719495). Whole blood, E1 (estrone) and E2 (estradiol) levels were collected at baseline (T1), 4 weeks (T2), 12 weeks (T3) and 24 weeks (T4) after ET initiation. 5 cohorts were enrolled: premenopausal and postmenopausal HCs, premenopausal pts on tamoxifen (TAM) or aromatase inhibitors (AIs) plus ovarian suppression, postmenopausal pts on AIs. Bulk RNA sequencing of whole blood was performed using NuGEN mRNA-seq on the Illumina Nova-seq platform. Differential expression analysis was performed using DESeq2 between HC and ET groups at T1, and between T1 and T4 within BC pts. The Benjamini-Hochberg method was used to adjust the P values for multiple comparisons. A curated set of 192 immune related gene signatures was used in the gene signature analysis. For each signature/module, a score was computed by taking the median of the expression values from all genes in each gene signature. CIBERSORT analysis was performed using the LM22 signature gene file and 100 permutations. Paired t-test was performed to compare T1 and T4. Results: Between 2019 and 2023, 64 pts were enrolled. Pts with missing samples or prior chemotherapy were excluded in this analysis. T1 samples of 25 pts with BC versus 14 HCs revealed marked changes in circulating immune cell (IC) composition with 537 DEGs (p value < 0.05). Circulating ICs in BC pts had marked upregulation of genes associated with oxidative phosphorylation, adipogenesis, interferon alpha, reactive oxygen species compared to HCs. Immune cell composition was inferred by CIBERSORT analysis which suggests that BC pts had fewer circulating naïve B cells (p=0.048) whereas regulatory T cells (p=0.025) and activated NK cells (p=0.0039) were higher in BC pts than HCs at T1. BC pts with reductions in E1/E2 levels 6 months after ET had 77 DEGs in PBMCs between T1 and T4. Gene signature analysis shows that B cells and NK cells (p=0.0077) signatures are elevated post ET whereas a reduction of neutrophil signature (p=0.0034) was noted. Conclusions: There are significant differences in the baseline IC repertoire/activity in BC pts compared to HCs. The reduction in B cell activity/number and an increase in regulatory T cell number suggests a possible compromised immune system in BC pts. After surgery and 6 months of ET, these suppressed immune phenotypes reversed. Clinical trial information: NCT03719495 .

Effects of immune cells in mediating the relationship between gut microbiota and myelodysplastic syndrome: a bidirectional two-sample, two-step Mendelian randomization study

BackgroundThe definitive establishment of a causal relationship between gut microbiota and myelodysplastic syndrome (MDS) has not been achieved. Furthermore, the involvement of immune cells in mediating the connection between gut microbiota and MDS is presently unclear.MethodsTo elucidate the bidirectional correlation between gut microbiota and MDS, as well as to investigate the mediating role of immune cells, a bidirectional two-sample, two-step Mendelian randomization (MR) study was conducted. Summary statistics were obtained from genome-wide association studies (GWAS), including MDS (456,348 individuals), gut microbiota (18,340 individuals), and 731 immune cells signatures (3757 individuals).ResultsGenetically predicted eight gut microbiota traits were significantly associated with MDS risk, but not vice versa. Through biological annotation of host-microbiome shared genes, we found that immune regulation may mediate the impact of gut microbiota on MDS. Subsequently, twenty-three immunophenotypes that exhibited significant associations with MDS risk and five of these immunophenotypes were under the causal influence of gut microbiota. Importantly, the causal effects of gut microbiota on MDS were significantly mediated by five immunophenotypes, including CD4 +T cell %leukocyte, CD127 on CD45RA − CD4 not regulatory T cell, CD45 on CD33 + HLA DR + WHR, CD33 on basophil, and Monocyte AC.ConclusionsGut microbiota was causally associated with MDS risk, and five specific immunophenotypes served as potential causal mediators of the effect of gut microbiota on MDS. Understanding the causality among gut microbiota, immune cells and MDS is critical in identifying potential targets for diagnosis and treatment.

Memory CD8 T cells are vulnerable to chronic IFN-γ signals but not to CD4 T cell deficiency in MHCII-deficient mice

The mechanisms by which the number of memory CD8 T cells is stably maintained remains incompletely understood. It has been postulated that maintaining them requires help from CD4 T cells, because adoptively transferred memory CD8 T cells persist poorly in MHC class II (MHCII)-deficient mice. Here we show that chronic interferon-γ signals, not CD4 T cell-deficiency, are responsible for their attrition in MHCII-deficient environments. Excess IFN-γ is produced primarily by endogenous colonic CD8 T cells in MHCII-deficient mice. IFN-γ neutralization restores the number of memory CD8 T cells in MHCII-deficient mice, whereas repeated IFN-γ administration or transduction of a gain-of-function STAT1 mutant reduces their number in wild-type mice. CD127high memory cells proliferate actively in response to IFN-γ signals, but are more susceptible to attrition than CD127low terminally differentiated effector memory cells. Furthermore, single-cell RNA-sequencing of memory CD8 T cells reveals proliferating cells that resemble short-lived, terminal effector cells and documents global downregulation of gene signatures of long-lived memory cells in MHCII-deficient environments. We propose that chronic IFN-γ signals deplete memory CD8 T cells by compromising their long-term survival and by diverting self-renewing CD127high cells toward terminal differentiation.

Impeller: a path-based heterogeneous graph learning method for spatial transcriptomic data imputation.

Recent advances in spatial transcriptomics allow spatially resolved gene expression measurements with cellular or even sub-cellular resolution, directly characterizing the complex spatiotemporal gene expression landscape and cell-to-cell interactions in their native microenvironments. Due to technology limitations, most spatial transcriptomic technologies still yield incomplete expression measurements with excessive missing values. Therefore, gene imputation is critical to filling in missing data, enhancing resolution, and improving overall interpretability. However, existing methods either require additional matched single-cell RNA-seq data, which is rarely available, or ignore spatial proximity or expression similarity information. To address these issues, we introduce Impeller, a path-based heterogeneous graph learning method for spatial transcriptomic data imputation. Impeller has two unique characteristics distinct from existing approaches. First, it builds a heterogeneous graph with two types of edges representing spatial proximity and expression similarity. Therefore, Impeller can simultaneously model smooth gene expression changes across spatial dimensions and capture similar gene expression signatures of faraway cells from the same type. Moreover, Impeller incorporates both short- and long-range cell-to-cell interactions (e.g. via paracrine and endocrine) by stacking multiple GNN layers. We use a learnable path operator in Impeller to avoid the over-smoothing issue of the traditional Laplacian matrices. Extensive experiments on diverse datasets from three popular platforms and two species demonstrate the superiority of Impeller over various state-of-the-art imputation methods. The code and preprocessed data used in this study are available at https://github.com/aicb-ZhangLabs/Impeller and https://zenodo.org/records/11212604.

Single-cell RNA-binding protein pattern-mediated molecular subtypes depict the hallmarks of the tumor microenvironment in bladder urothelial carcinoma

Abstract Objectives Bladder carcinoma (BC) is a common malignancy of the urinary tract. As a new hallmark of cancer for drug therapy, RNA-binding proteins (RBPs) are key regulatory factors in alternative splicing events. This work is to uncover the relationship between BC and RBP in order to find drug targets in BC. Methods In this work, data from single-cell RNA-seq GSE1355337, PRJNA662018, and the TCGA-Bladder urothelial carcinoma (BLCA) cohorts are integrated to identify their relationships. A scoring system is constructed according to RBPs gene expression and patients’ survival. A network is constructed to analyze the alternative splicing events and RBP genes. Results A scoring system identified 321 RBPs significantly associated with the prognosis of patients. Subsequent typing of these RBP genes in two single-cell datasets demonstrated that most of the RBP genes had variable copy numbers. Three RBP clusters were identified. Using RBP genes as a signature in BC epithelial cells allows for differentiation between different grades of BC samples. The novel RBP genes-based subtype system reflects BC clinical staging. Notably, CellChat analysis revealed that the RBP genes-associated cell subtypes of T cells had extensive interactions with epithelial cells. Further analysis showed that the ligand-receptor pair MIF-CXCR4 mediated the communication between RBP-associated subtypes of BC epithelial cells and T cells. Conclusions Taken together, RBP genes are associated with BC progress and offer new indicators for precision medicine in BC.

Causal role of immune cells in hypertension: a bidirectional Mendelian randomization study.

Although Hypertension (HTN) is considered to be a cardiovascular disease caused by multiple factors, the cause of it is still unknown. In this study, we aim to find out whether circulating immune cell characteristics have an impact on susceptibility to HTN. This study employed a comprehensive two-sample Mendelian randomization (MR) analysis to investigate the causal association between immune cell characteristics and HTN. Utilizing publicly accessible genetic data, we examined the causal relationship between HTN and the susceptibility to 731 immune cell signatures. To ensure the reliability and validity of the findings, a comprehensive sensitivity analysis was conducted to assess heterogeneity, confirm the robustness of the results and evaluate the presence of horizontal pleiotropy. After FDR correction, immune phenotype had an effect on HTN. In our study, one immunophenotype was identified as being positively associated with HTN risk significance: HLA DR on CD33- HLA DR+. In addition, we examined 8 immune phenotype with no statistically significant effect of HTN, but it is worth mentioning that they had an unadjusted low P-value phenotype. Our MR study by genetic means demonstrated the close relationship between HTN and immune cells, thus providing guidance for future clinical prediction and subsequent treatment of HTN.

Adenosine 5'-Monophosphate-to-Threonine Ratio Promotes Abdominal Aortic Aneurysms via Up-Regulation of HLA-DR on Natural Killer Cells: A Bidirectional Mendelian Randomized Analysis.

Objective: Immune-metabolic interactions may have causal and therapeutic impacts on abdominal aortic aneurysms (AAAs). However, due to the lack of research on the relationship between immune-metabolic interactions and AAAs, further exploration of the mechanism faces challenges. Methods: A two-sample, two-step mediation analysis with Mendelian randomization (MR) based on genome-wide association studies (GWASs) was performed to determine the causal associations among blood immune cell signatures, metabolites, and AAAs. The stability, heterogeneity, and pleiotropy of the results were verified using a multivariate sensitivity analysis. Results: After multiple two-sample MRs using the AAA data from two large-scale GWAS databases, we determined that the human leukocyte antigen-DR (HLA-DR) levels on HLA-DR + natural killer (NK) cells (HLA-DR/NK) were associated with the causal effect of an AAA, with consistent results in the two databases (FinnGen: odds ratio (OR) = 1.054, 95% confidence interval (CI): 1.003-1.067, p-value = 0.036; UK Biobank: OR = 1.149, 95% CI: 1.046-1.261, p-value = 0.004). The metabolites associated with the risk of developing an AAA were enriched to find a specific metabolic model. We also found that the ratio of adenosine 5'-monophosphate (AMP) to threonine could act as a potential mediator between the HLA/NK and an AAA, with a direct effect (beta effect = 0.0496) and an indirect effect (beta effect = 0.0029). The mediation proportion was 5.56%. Conclusions: Our study found that an up-regulation of HLA-DR on HLA-DR/NK cells can increase the risk of an AAA via improvements in the AMP-to-threonine ratio, thus providing a potential new biomarker for the prediction and treatment of AAAs.

Spatial transcriptomic brain imaging reveals the effects of immunomodulation therapy on specific regional brain cells in a mouse dementia model

Increasing evidence of brain-immune crosstalk raises expectations for the efficacy of novel immunotherapies in Alzheimer’s disease (AD), but the lack of methods to examine brain tissues makes it difficult to evaluate therapeutics. Here, we investigated the changes in spatial transcriptomic signatures and brain cell types using the 10x Genomics Visium platform in immune-modulated AD models after various treatments. To proceed with an analysis suitable for barcode-based spatial transcriptomics, we first organized a workflow for segmentation of neuroanatomical regions, establishment of appropriate gene combinations, and comprehensive review of altered brain cell signatures. Ultimately, we investigated spatial transcriptomic changes following administration of immunomodulators, NK cell supplements and an anti-CD4 antibody, which ameliorated behavior impairment, and designated brain cells and regions showing probable associations with behavior changes. We provided the customized analytic pipeline into an application named STquantool. Thus, we anticipate that our approach can help researchers interpret the real action of drug candidates by simultaneously investigating the dynamics of all transcripts for the development of novel AD therapeutics.

Harnessing genetic interactions for prediction of immune checkpoint inhibitors response signature in cancer cells

Genetic interactions (GIs) refer to two altered genes having a combined effect that is not seen individually. They play a crucial role in influencing drug efficacy. We utilized CGIdb 2.0 (http://www.medsysbio.org/CGIdb2/), an updated database of comprehensively published GIs information, encompassing synthetic lethality (SL), synthetic viability (SV), and chemical-genetic interactions. CGIdb 2.0 elucidates GIs relationships between or within protein complex models by integrating protein-protein physical interactions. Additionally, we introduced GENIUS (GENetic Interactions mediated drUg Signature) to leverage GIs for identifying the response signature of immune checkpoint inhibitors (ICIs). GENIUS identified high MAP4K4 expression as a resistant signature and high HERC4 expression as a sensitive signature for ICIs treatment. Melanoma patients with high expression of MAP4K4 were associated with decreased efficacy and poorer survival following ICIs treatment. Conversely, overexpression of HERC4 in melanoma patients correlated with a positive response to ICIs. Notably, HERC4 enhances sensitivity to immunotherapy by facilitating antigen presentation. Analyses of immune cell infiltration and single-cell data revealed that B cells expressing MAP4K4 may contribute to resistance to ICIs in melanoma. Overall, CGIdb 2.0, provides integrated GIs data, thus serving as a crucial tool for exploring drug effects.

#1154 Type I interferon pathway is upregulated in microscopic polyangiitis (vs granulomatosis with polyangiitis)

Abstract Background and Aims The two main types of ANCA-associated vasculitis (AAV), microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA), share common characteristics but also differ on many aspects (epidemiological, genetic, clinical, outcomes). These differences did not justify, to date, different patient management. To identify more specific targetable pathways, we characterized the kidney transcriptome of MPA vs. GPA patients with AAV-glomerulonephritis (AAV-GN). Method This retrospective study included adult patients with AAV-GN from the French Maine-Anjou Registry. Immune gene transcript analysis was performed on RNA extracted from 97 kidney biopsies using NanoString technology. Significant transcripts were examined to identify immune pathways of interest. We then used a publicly available dataset to explore these identified pathways (qPCR / ELISA). Results We compared kidney biopsies from GPA (n = 33) and MPA (n = 64) patients. There was no difference in eGFR between both groups at diagnosis. Of the 750 evaluated immune transcripts, 8 genes were found differentially expressed between MPA and GPA patients, all being upregulated in MPA (log2FC > 1, q-value < 0.05) (Fig. 1A). Seven (out of 8) genes belonged to the type I interferon (IFN-I) signaling pathway. A personal analysis of unpublished, publicly available, data1 identified a significantly more pronounced IFN-I signature in the peripheral blood mononuclear cells of patients with MPO-AAV when compared to those with PR3-AAV (Fig. 1B). Serum levels or CXCL10, a cytokine that mediates immune responses through the recruitment and activation of numerous immune cells under the regulation of IFN-I, were found higher in MPO-AAV patients (Fig. 1C). Conclusion We identified a type I interferon signature, both in kidneys and in blood, in patients with MPA (or MPO-AAV) in comparison to GPA (or PR3-AAV). This is in line with previous lines of evidence brough by Kessenbrock et al. and Kessler et al. suggesting a role for IFN-I in AAV and especially in MPO-AAV. This signature may help gain a deeper understanding of the AAV-GN pathogenesis and provide insights for developing new therapeutic options.

#1955 Ferroptotic proteins ACSL4 and ALOX15 as therapeutic targets in AKI

Abstract Background and Aims Ferroptosis is a regulated form of necrosis which is dependent on cellular iron and is characterized by the accumulation of lipid peroxides and failure of cellular antioxidant defences. We have previously described in vivo that ferroptosis is the primary cause of folic acid-induced acute kidney injury (FA-AKI) and that necroinflammation secondary to ferroptosis may further worsen kidney injury, since ferroptosis inhibition improved kidney function and decreased tubular cell death and oxidative stress. Acyl-CoA synthetase long-chain family member 4 (ACSL4) is involved in the incorporation of polyunsaturated fatty acid (PUFA) into membranes, while 15-Lipoxygenase (Alox15) catalyzes the regio- and enantioselective peroxidation of membrane-esterified PUFAs, forming the ultimate peroxidized species that induce ferroptosis. In the present work, we aim to explore the potential of both proteins as therapeutic targets in AKI, as well as the molecular characterization of kidney ferroptosis in cultured tubular cells. Method Animal model: Female 12- to 14-week-old C57BL/6J wild type mice received a single intraperitoneal (i.p) injection of folic acid or vehicle and were sacrificed 48 hours later. Troglitazone was intravenously administered for in vivo ACSL4 inhibition, and Compound 1 was i.p administered for in vivo ALOX15 inhibition. In vitro characterization of ferroptosis: HK2 human tubular kidney cells were incubated with arachidonic acid (AA) prior to sublethal RSL3 administration for sensitization to ferroptosis. We used Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lpx-1) as inhibitors of ferroptosis. The thiazolidinedione family members Rosiglitazone, Pioglitazone and Troglitazone were used as pharmacological inhibitors of ACSL4. We also performed transcriptional silencing of ACSL4 using a specific siRNA. Compound 1 and PD1646 were administered for in vitro Alox15 inhibition. Cellular lipid peroxidation was analyzed by flow cytometry with Bodipy 581/591 staining. To characterize the lipid signature of ferroptotic cells, supernatants were collected and 15-HETEs levels were measured by ELISA. Oxido-lipidomic analysis of ferroptotic cells was performed by LC-MS. Real Time PCR and Western Blot of whole kidneys and cultured cells were performed for the detection and quantification of ACSL4 and ALOX15. Cell death was assessed by measurement of cell viability (MTT assay) and cytotoxicity (LDH assay). Tissue cell death was assessed by TUNEL. Results Kidney transcriptomics identified Acsl4 as the most upregulated member of Acsl family during FA-AKI. This was validated at mRNA and protein levels. Supplementation with AA, the preferred ACSL4 substrate, sensitized HK2 tubular cells to ferroptosis under sublethal RSL3 conditions, and this was prevented with specific ferroptosis inhibitors Fer-1 and Lpx-1. Pharmacological inhibition of ACSL4 with Troglitazone and a specific siRNA protected from cell death and lipid peroxidation induced by co-stimulation of AA and RSL3 in HK2 cells. Likewise, ALOX15 proteins levels were also increased in FA-AKI at 48 hours. PD1646 and Compound 1, both used to inhibit ALOX15, protected from ferroptotic cell death and lipid peroxidation in HK2 cells. Lipidomic analysis on HK2 tubular cells stimulated with AA+RSL3 uncovered an increased content of key peroxidized lipid species involved in ferroptosis execution, which was alleviated by targeting ACSL4 or ALOX15. Additionally, renal function of FA-AKI in mice was improved by ACSL4 pharmacological inhibition with Troglitazone and with ALOX15 inhibition with Compound 1. Conclusion Our preliminary results suggest therapeutic potential of ACSL4 and ALOX15 as ferroptotic targes during AKI. High PUFA content sensitizes human tubular HK2 cells to cell death and lipid peroxidation in the presence of sublethal ferroptotic triggers, and this was prevented by targeting ACSL4 or ALOX15 both in vitro and in vivo in FA-AKI. Closely related lipid peroxide species were decreased by ACSL4 or ALOX15 inhibitors.

#1511 The immune cell ‘signature’ of incident hypertension

Abstract Background and Aims Hypertension results from complex genetic and environmental interactions, mediated by cardiac, vascular, endocrine, and renal systems. The immune system may modulate all of these, though the exact mechanisms linking immune activation with hypertension and organ damage remain poorly defined. We aimed to identify the immune cell ‘signature’ in patients with newly diagnosed, untreated hypertension (HTN; n=76) compared to normotensive controls (NTN; n=67), as well as in relation to nocturnal dipping status. Method Flow cytometry of peripheral blood mononuclear cells (Attune, Thermo Fisher) characterising B cell, T cell, monocyte and dendritic cells (DC) subsets, including activation signature with intracellular staining, in an exploratory comparative analysis. Results The proportion of CD4+ naïve T cells (CD45RA+CCR7+) was lower in hypertension, with CD45RO+CD62L+ cells attaining nominal statistical significance between NTN and HTN groups (1.3 ± 1.1 vs 1.2 ± 1.0, p=0.039). CD4+ T central memory cells were expanded (TCM, 23.1 ± 7.2 vs 27.7 ± 10.0, p=0.002, Figure) in HTN along with T effector memory (TEM) cells lacking CD62 in an adjusted model, (TEM, CD4+CD45RA-CCR7-CD62l-; 31.4 ± 7.5 vs 33.7 ± 10.3, p0.035; CD8+CD45RA-CCR7-CD45RO+CD62l− 48.9 ± 10.5 vs 52.2 ± 13.3, p=0.011). Hypertensive group had proportionally fewer CD4+CD28+ cells (95.1 ± 9.8 vs 92.0 ± 13.5, p=0.034) and CD8+CD28+ (68.0 ± 19.7 vs 62.0 ± 21.0, p=0.011), as well as CD8+ TEMRA cells (Figure). In a fully adjusted model, the CCR7-CD45RO-CD62L+ (52.2 ± 14.5 vs 50.2 ± 16.1, p=0.036), and CD8+CD28+CD45RA+CCR7- significantly differed between groups (23.1 ± 14.7 vs 25.0 ± 17.8, p=0.008). On the molecular level, T cells were polarised in HTN towards a Th1/Tc1 and Th17.1/Tc17.1 pro-inflammatory milieu (Figure). No differences were detected in B cell subsets. The innate immune ‘signature’ was characterised by intermediate monocytes with a differing pattern of CCR2 and CCR5 chemokine receptor expression (Table 1), and alterations in STAT1 and STAT6 phosphorylation cascades. NK cell CD56+Dim expression and fewer CD122-expressing NKT (0.13 ± 0.0 vs 0.08 ± 0.1, p=0.046) and CD122+ T lymphocytes (1.3 ± 3.4 vs 0.6 ± 0.4, p=0.019) further supported pro-inflammatory milieu in hypertension. Finally, in an exploratory nominal statistical analysis, nocturnal non-dipping was associated with particular changes in immune cell signature, including expanded CD4+TCM (CD4+CD45RO+CD62L-, 9.4 ± 4.3 vs 8.0 ± 3.3, p=0.04) and reduced CD4+TEM and TEMRA compartments; polarisation towards Th17 and Tc1; and altered Monocyte CCR2 and CCR5 expression. Additionally, non-dipping was linked to dendritic cell mannose receptor downregulation e.g. mDC non-dipper 10.9 ± 5.8 vs dipper 13.6 ± 9.7, p=0.03. Conclusion These exploratory data support alterations of circulating innate (monocytes) and adaptive (T cells) immune compartments in newly diagnosed untreated, and uncomplicated hypertension.

Key Roles of CACNA1C/Cav1.2 and CALB1/Calbindin in Prefrontal Neurons Altered in Cognitive Disorders.

The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by β1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or β1-adrenoceptor antagonist protected working memory from stress. The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.

BET inhibition reforms the immune microenvironment and alleviates T cell dysfunction in chronic lymphocytic leukemia.

Redundant tumor microenvironment (TME) immunosuppressive mechanisms and epigenetic maintenance of terminal T cell exhaustion greatly hinder functional antitumor immune responses in chronic lymphocytic leukemia (CLL). Bromodomain and extraterminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T cell function and differentiation. Herein, we report that blocking BET protein function alleviates immunosuppressive networks in the CLL TME and repairs inherent CLL T cell defects. The pan-BET inhibitor OPN-51107 reduced exhaustion-associated cell signatures resulting in improved T cell proliferation and effector function in the Eμ-TCL1 splenic TME. Following BET inhibition (BET-i), TME T cells coexpressed significantly fewer inhibitory receptors (IRs) (e.g., PD-1, CD160, CD244, LAG3, VISTA). Complementary results were witnessed in primary CLL cultures, wherein OPN-51107 exerted proinflammatory effects on T cells, regardless of leukemic cell burden. BET-i additionally promotes a progenitor T cell phenotype through reduced expression of transcription factors that maintain terminal differentiation and increased expression of TCF-1, at least in part through altered chromatin accessibility. Moreover, direct T cell effects of BET-i were unmatched by common targeted therapies in CLL. This study demonstrates the immunomodulatory action of BET-i on CLL T cells and supports the inclusion of BET inhibitors in the management of CLL to alleviate terminal T cell dysfunction and potentially enhance tumoricidal T cell activity.

Lack of TGFβ signaling competency predicts immune poor cancer conversion to immune rich and response to checkpoint blockade.

Transforming growth factor beta (TGFβ) is well-recognized as an immunosuppressive player in the tumor microenvironment but also has a significant impact on cancer cell phenotypes. Loss of TGFβ signaling impairs DNA repair competency, which is described by a transcriptomic score, βAlt. Cancers with high βAlt have more genomic damage and are more responsive to genotoxic therapy. The growing appreciation that cancer DNA repair deficits are important determinants of immune response prompted us to investigate the association of βAlt with response to immune checkpoint blockade (ICB). We predicted that high βAlt tumors would be infiltrated with lymphocytes because of DNA damage burden and hence responsive to ICB. We analyzed public transcriptomic data from clinical trials and preclinical models using transcriptomic signatures of TGFβ targets, DNA repair genes, tumor educated immune cells and interferon. A high βAlt, immune poor mammary tumor derived transplant model resistant to programmed death ligand 1 (PD-L1) antibodies was studied using multispectral flow cytometry to interrogate the immune system. Metastatic bladder patients in IMvigor 210 who responded to ICB had significantly increased βAlt scores and experienced significantly longer overall survival compared to those with low βAlt scores (hazard ratio 0.62, P=0.011) . Unexpectedly, 75% of high βAlt cancers were immune poor as defined by low expression of tumor educated immune cell and interferon signatures. The association of high βAlt with immune poor cancer was also evident in TCGA and preclinical cancer models. We used a high βAlt, immune poor cancer to test therapeutic strategies to overcome its inherent anti-PD-L1 resistance. Combination treatment with radiation and TGFβ inhibition were necessary for lymphocytic infiltration and activated NK cells were required for ICB response. Bioinformatic analysis identified high βAlt, immune poor B16 and CT26 preclinical models and paired biopsies of cancer patients that also demonstrated NK cell activation upon response to ICB. Our studies support βAlt as a biomarker that predicts response to ICB albeit in immune poor cancers, which has implications for the development of therapeutic strategies to increase the number of cancer patients who will benefit from immunotherapy.

Relationship between immune cells and diabetic nephropathy: a Mendelian randomization study.

Although previous studies have suggested potential correlations between immunocytes and diabetic nephropathy (DN), the causal correlations between them remain unclarified. In this study, we employed Mendelian randomization (MR) to analyze the potential causative correlations between immune 731 cells and DN. We used the Genome-Wide Association Studies (GWAS) database to aggregate signatures of immune cells and DN from European and East Asian populations. Single-nucleotide polymorphisms (SNPs) were used as instrumental variables. MR analysis was conducted using Mendelian randomization-Egger (MR-Egger) regression and the random-effects inverse-variance weighted (IVW) method. A total of 3,571 SNPs were included as instrumental variables. The MR-Egger regression model indicated no genetic pleiotropy (P = 0.6284). The results of the IVW method indicated a statistically significant causal relationship between immune cell HLA-DR on CD14-CD16- (P = 0.029), CD45RA-CD28-CD8 + T cell% T cells (P = 0.0278), CD11c on myeloid dendritic cells (P = 0.0352), HLA-DR on CD14+ monocytes (P < 0.001), CD27 on CD24 + CD27 + B cells (P = 0.0334), CD27 on IgD + CD24 + B cells (P = 0.0137), CD4 on CD39 + CD4 + T cells (P = 0.0347), CD28 on CD39 + CD4 + T cells (P = 0.0414), CD39 on CD39 + CD4 + T cells (P = 0.0426), and DN. Additionally, there was no heterogeneity in SNPs related HLA-DR on CD14-CD16-cells and DN(I2 = 32%, Cochran's Q = 2.9476, P = 0.2291). Moreover, leave-one-out analysis showed a causal correlation between HLA-DR on CD14-CD16- cells and DN. Higher expression of immune cell HLA-DR on CD14-CD16- cells may indicate a lower risk of DN.

Preoperative neoadjuvant targeted therapy remodels intra-tumoral heterogeneity of clear-cell renal cell carcinoma and ferroptosis inhibition induces resistance progression

Neoadjuvant tyrosine kinase inhibitor (TKI) therapy is an important treatment option for advanced renal cell carcinoma (RCC). Many RCC patients may fail to respond or be resistant to TKI therapy. We aimed to explore the key mechanisms of neoadjuvant therapy résistance. We obtained tumor samples from matched pre-treatment biopsy and post-treatment surgical samples and performed single-cell RNA sequencing. Sunitinib-resistant ccRCC cell lines were established. Ferroptosis was detected by ferrous ion and lipid peroxidation levels. Tumor growth and resistance to Sunitinib was validated in vitro and vivo. Immunohistochemistry was used to validate the levels key genes and lipid peroxidation. Multi-center cohorts were included, including TCGA, ICGC, Checkmate-025 and IMmotion151 clinical trial. Survival analysis was performed to identify the associated clinical and genomic variables. Intratumoral heterogeneity was first described in the whole neoadjuvant management. The signature of endothelial cells was correlated with drug sensitivity and progression-free survival. Ferroptosis was shown to be the key biological program in malignant cell resistance. We observed tissue lipid peroxidation was negatively correlated with IL6 and tumor response. TKI-resistant cell line was established. SLC7A11 knockdown promoted cell growth and lipid peroxidation, increased the ferroptosis level, and suppressed the growth of tumor xenografts significantly (P < 0.01). IL6 could reverse the ferroptosis and malignant behavior caused by SLC7A11 (−) via JAK2/STAT3 pathway, which was rescued by the ferroptosis inducer Erastin. Our data indicate that ferroptosis is a novel strategy for advanced RCC treatment, which activated by IL6, providing a new idea for resistance to TKIs.

Profiling of Early Immune Responses to Vaccination Using THP-1-Derived Dendritic Cells.

The COVID-19 pandemic has made assessing vaccine efficacy more challenging. Besides neutralizing antibody assays, systems vaccinology studies use omics technology to reveal immune response mechanisms and identify gene signatures in human peripheral blood mononuclear cells (PBMCs). However, due to their low proportion in PBMCs, profiling the immune response signatures of dendritic cells (DCs) is difficult. Here, we develop a predictive model for evaluating early immune responses in dendritic cells. We establish a THP-1-derived dendritic cell (TDDC) model and stimulate their maturation in vitro with an optimal dose of attenuated yellow fever 17D (YF-17D). Transcriptomic analysis reveals that type I interferon (IFN-I)-induced immunity plays a key role in dendritic cells. IFN-I regulatory biomarkers (IRF7, SIGLEC1) and IFN-I-inducible biomarkers (IFI27, IFI44, IFIT1, IFIT3, ISG15, MX1, OAS2, OAS3) are identified and validated in vitro and in vivo. Furthermore, we apply this TDDC approach to various types of vaccines, providing novel insights into their early immune response signatures and their heterogeneity in vaccine recipients. Our findings suggest that a standardizable TDDC model is a promising predictive approach to assessing early immunity in DCs. Further research into vaccine efficacy assessment approaches on various types of immune cells could lead to a systemic regimen for vaccine development in the future.

A prognostic neural epigenetic signature in high-grade glioma

Neural–tumor interactions drive glioma growth as evidenced in preclinical models, but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients’ survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma, primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like, astrocyte-like and oligodendrocyte-progenitor-like, alongside oligodendrocytes and excitatory neurons. In line with these findings, high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients, a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients’ plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes.

Impaired signaling pathways on Berardinelli-Seip congenital lipodystrophy macrophages during Leishmania infantum infection.

Berardinelli-Seip congenital lipodystrophy (CGL), a rare autosomal recessive disorder, is characterized by a lack of adipose tissue. Infections are one of the major causes of CGL individuals' premature death. The mechanisms that predispose to infections are poorly understood. We used Leishmania infantum as an in vitro model of intracellular infection to explore mechanisms underlying the CGL infection processes, and to understand the impact of host mutations on Leishmania survival, since this pathogen enters macrophages through specialized membrane lipid domains. The transcriptomic profiles of both uninfected and infected monocyte-derived macrophages (MDMs) from CGL (types 1 and 2) and controls were studied. MDMs infected with L. infantum showed significantly downregulated expression of genes associated with infection-response pathways (MHC-I, TCR-CD3, and granzymes). There was a transcriptomic signature in CGL cells associated with impaired membrane trafficking and signaling in response to infection, with concomitant changes in the expression of membrane-associated genes in parasites (e.g. δ-amastins). We identified pathways suggesting the lipid storage dysfunction led to changes in phospholipids expression and impaired responses to infection, including immune synapse (antigen presentation, IFN-γ signaling, JAK/STAT); endocytosis; NF-kappaB signaling; and phosphatidylinositol biosynthesis. In summary, lipid metabolism of the host plays an important role in determining antigen presentation pathways.