Although most patients with type 1 diabetes (T1D) retain some functional insulin-producing islet β cells at the time of diagnosis, the rate of further β cell loss varies across individuals. It is not ...clear what drives this differential progression rate. CD8+ T cells have been implicated in the autoimmune destruction of β cells. Here, we addressed whether the phenotype and function of autoreactive CD8+ T cells influence disease progression. We identified islet-specific CD8+ T cells using high-content, single-cell mass cytometry in combination with peptide-loaded MHC tetramer staining. We applied a new analytical method, DISCOV-R, to characterize these rare subsets. Autoreactive T cells were phenotypically heterogeneous, and their phenotype differed by rate of disease progression. Activated islet-specific CD8+ memory T cells were prevalent in subjects with T1D who experienced rapid loss of C-peptide; in contrast, slow disease progression was associated with an exhaustion-like profile, with expression of multiple inhibitory receptors, limited cytokine production, and reduced proliferative capacity. This relationship between properties of autoreactive CD8+ T cells and the rate of T1D disease progression after onset make these phenotypes attractive putative biomarkers of disease trajectory and treatment response and reveal potential targets for therapeutic intervention.
Voltage-sensitive phosphatases (VSPs) are proteins that directly couple changes in membrane electrical potential to inositol lipid phosphatase activity. VSPs thus couple two signaling pathways that ...are critical for cellular functioning. Although a number of nonmammalian VSPs have been characterized biophysically, mammalian VSPs are less well understood at both the physiological and biophysical levels. In this study, we aimed to address this gap in knowledge by determining whether the VSP from mouse, Mm-VSP, is expressed in the brain and contains a functional voltage-sensing domain (VSD) and a phosphatase domain. We report that Mm-VSP is expressed in neurons and is developmentally regulated. To address whether the functions of the VSD and phosphatase domain are retained in Mm-VSP, we took advantage of the modular nature of these domains and expressed each independently as a chimeric protein in a heterologous expression system. We found that the Mm-VSP VSD, fused to a viral potassium channel, was able to drive voltage-dependent gating of the channel pore. The Mm-VSP phosphatase domain, fused to the VSD of a nonmammalian VSP, was also functional: activation resulted in PI(4,5)P2 depletion that was sufficient to inhibit the PI(4,5)P2-regulated KCNQ2/3 channels. While testing the functionality of the VSD and phosphatase domain, we observed slight differences between the activities of Mm-VSP-based chimeras and those of nonmammalian VSPs. Although the properties of VSP chimeras may not completely reflect the properties of native VSPs, the differences we observed in voltage-sensing and phosphatase activity provide a starting point for future experiments to investigate the function of Mm-VSP and other mammalian VSPs. In conclusion, our data reveal that both the VSD and the lipid phosphatase domain of Mm-VSP are functional, indicating that Mm-VSP likely plays an important role in mouse neurophysiology.
Black and Hispanic children growing up in disadvantaged urban neighborhoods have the highest rates of asthma and related morbidity in the United States.
This study sought to identify specific ...respiratory phenotypes of health and disease in this population, associations with early life exposures, and molecular patterns of gene expression in nasal epithelial cells that underlie clinical disease.
The study population consisted of 442 high-risk urban children who had repeated assessments of wheezing, allergen-specific IgE, and lung function through 10 years of age. Phenotypes were identified by developing temporal trajectories for these data, and then compared to early life exposures and patterns of nasal epithelial gene expression at 11 years of age.
Of the 6 identified respiratory phenotypes, a high wheeze, high atopy, low lung function group had the greatest respiratory morbidity. In early life, this group had low exposure to common allergens and high exposure to ergosterol in house dust. While all high-atopy groups were associated with increased expression of a type-2 inflammation gene module in nasal epithelial samples, an epithelium IL-13 response module tracked closely with impaired lung function, and a MUC5AC hypersecretion module was uniquely upregulated in the high wheeze, high atopy, low lung function group. In contrast, a medium wheeze, low atopy group showed altered expression of modules of epithelial integrity, epithelial injury, and antioxidant pathways.
In the first decade of life, high-risk urban children develop distinct phenotypes of respiratory health versus disease that link early life environmental exposures to childhood allergic sensitization and asthma. Moreover, unique patterns of airway gene expression demonstrate how specific molecular pathways underlie distinct respiratory phenotypes, including allergic and nonallergic asthma.
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Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ...ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5' position of the phosphoinositide.
Mucus plugging can worsen asthma control, lead to reduced lung function and fatal exacerbations. MUC5AC is the secretory mucin implicated in mucus plugging, and MUC5AC gene expression has been ...associated with development of airway obstruction and asthma exacerbations in urban children with asthma. However, the genetic determinants of MUC5AC expression are not established.
This study sought to assess single-nucleotide polymorphisms (SNPs) that influence MUC5AC expression and relate to pulmonary functions in childhood asthma.
This study used RNA-sequencing data from upper airway samples and performed cis-expression quantitative trait loci (eQTL) and allele-specific expression analyses in 2 cohorts of predominantly Black and Hispanic urban children, a high asthma-risk birth cohort, and an exacerbation-prone asthma cohort. Inducible MUC5AC eQTLs were further investigated during incipient asthma exacerbations. Significant eQTLs SNPs were tested for associations with lung function measurements and their functional consequences were investigated in DNA regulatory databases.
Two independent groups of SNPs in the MUC5AC gene that were significantly associated with MUC5AC expression were identified. Moreover, these SNPs showed stronger eQTL associations with MUC5AC expression during asthma exacerbations, which is consistent with inducible expression. SNPs in 1 group also showed significant association with decreased pulmonary functions. These SNPs included multiple EGR1 transcription factor binding sites, suggesting a mechanism of effect.
These findings demonstrate the applicability of organ-specific RNA-sequencing data to determine genetic factors contributing to a key disease pathway. Specifically, they suggest important genetic variations that may underlie propensity to mucus plugging in asthma and could be important in targeted asthma phenotyping and disease management strategies.
Genome-wide association studies of asthma have revealed robust associations with variation across the human leukocyte antigen (HLA) complex with independent associations in the HLA class I and class ...II regions for both childhood-onset asthma (COA) and adult-onset asthma (AOA). However, the specific variants and genes contributing to risk are unknown.
We used Bayesian approaches to perform genetic fine-mapping for COA and AOA (n=9432 and 21,556, respectively; n=318,167 shared controls) in White British individuals from the UK Biobank and to perform expression quantitative trait locus (eQTL) fine-mapping in immune (lymphoblastoid cell lines, n=398; peripheral blood mononuclear cells, n=132) and airway (nasal epithelial cells, n=188) cells from ethnically diverse individuals. We also examined putatively causal protein coding variation from protein crystal structures and conducted replication studies in independent multi-ethnic cohorts from the UK Biobank (COA n=1686; AOA n=3666; controls n=56,063).
Genetic fine-mapping revealed both shared and distinct causal variation between COA and AOA in the class I region but only distinct causal variation in the class II region. Both gene expression levels and amino acid variation contributed to risk. Our results from eQTL fine-mapping and amino acid visualization suggested that the HLA-DQA1*03:01 allele and variation associated with expression of the nonclassical HLA-DQA2 and HLA-DQB2 genes accounted entirely for the most significant association with AOA in GWAS. Our studies also suggested a potentially prominent role for HLA-C protein coding variation in the class I region in COA. We replicated putatively causal variant associations in a multi-ethnic cohort.
We highlight roles for both gene expression and protein coding variation in asthma risk and identified putatively causal variation and genes in the HLA region. A convergence of genomic, transcriptional, and protein coding evidence implicates the HLA-DQA2 and HLA-DQB2 genes and HLA-DQA1*03:01 allele in AOA.
Variation in the preservation of β cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response. The T1DAL trial targeted T ...cells with alefacept (LFA-3-Ig) and demonstrated C-peptide preservation in approximately 30% of new-onset T1D individuals. We analyzed islet antigen-reactive (IAR) CD4+ T cells in PBMC samples collected prior to treatment from alefacept- and placebo-treated individuals using flow cytometry and single-cell RNA sequencing. IAR CD4+ T cells at baseline had heterogeneous phenotypes. Transcript profiles formed phenotypic clusters of cells along a trajectory based on increasing maturation and activation, and T cell receptor (TCR) chains showed clonal expansion. Notably, the frequency of IAR CD4+ T cells with a memory phenotype and a unique transcript profile (cluster 3) were inversely correlated with C-peptide preservation in alefacept-treated, but not placebo-treated, individuals. Cluster 3 cells had a proinflammatory phenotype characterized by expression of the transcription factor BHLHE40 and the cytokines GM-CSF and TNF-α, and shared TCR chains with effector memory-like clusters. Our results suggest IAR CD4+ T cells as a potential baseline biomarker of response to therapies targeting the CD2 pathway and warrant investigation for other T cell-related therapies.
Clinical trials of biologic therapies in type 1 diabetes (T1D) aim to mitigate autoimmune destruction of pancreatic β cells through immune perturbation and serve as resources to elucidate ...immunological mechanisms in health and disease. In the T1DAL trial of alefacept (LFA3-Ig) in recent-onset T1D, endogenous insulin production was preserved in 30% of subjects for 2 years after therapy. Given our previous findings linking exhausted-like CD8+ T cells to beneficial response in T1D trials, we applied unbiased analyses to sorted CD8+ T cells to evaluate their potential role in T1DAL. Using RNA sequencing, we found that greater insulin C-peptide preservation was associated with a module of activation- and exhaustion-associated genes. This signature was dissected into 2 CD8 memory phenotypes through correlation with cytometry data. These cells were hypoproliferative, shared expanded rearranged TCR junctions, and expressed exhaustion-associated markers including TIGIT and KLRG1. The 2 phenotypes could be distinguished by reciprocal expression of CD8+ T and NK cell markers (GZMB, CD57, and inhibitory killer cell immunoglobulin-like receptor iKIR genes), versus T cell activation and differentiation markers (PD-1 and CD28). These findings support previous evidence linking exhausted-like CD8+ T cells to successful immune interventions for T1D, while suggesting that multiple inhibitory mechanisms can promote this beneficial cell state.
BackgroundEstablished biomarkers of immune checkpoint inhibitor (ICI) response in metastatic non-small cell lung cancer (mNSCLC), such as PD-L1 and tumor mutational burden (TMB), do not identify all ...patients with durable response. While biomarkers spanning multiple data modalities have been proposed to address this unmet need, additional evidence is required for use in the clinic. Here, we performed a comparative study assessing the association between previously described biomarkers of ICI response and outcomes in a real-world mNSCLC cohort.MethodsUsing the Tempus database, we analyzed de-identified records of non-squamous EGFR-negative and ALK fusion-negative mNSCLC patients treated with first-line ICI regimens and profiled with targeted-panel DNA-seq and whole-exome-capture RNA-seq. ICI-related biomarkers were calculated following published methods using the Tempus IO platform (table 1). Immune biomarkers included features describing tumor biology (including PD-L1 IHC, STK11 and KEAP1 mutations, APOBEC signature1), HLA (HLA-LOH, HLA A*03 genotype), neoantigens (TMB, neoantigen burden), and RNA-based signatures of immune activity such as IFNγ tumor inflammation signature,2 IMPRES,3 Cytotoxic score,4 NRS,5tertiary lymphoid structure (TLS),6 7 and others. Real-world time to progression (rwTTP) was defined as the interval from ICI start to the first progression event, censored on last known physician encounter. Cox proportional hazards (Cox PH) models were fitted to evaluate the relationship between each biomarker and rwTTP.ResultsIn this mNSCLC cohort (N=199), multiple RNA-based signatures related to immune infiltration, TLS, cytotoxic and cytolytic activity, and IFNγ tumor inflammation signature (TIS) were significantly associated with longer rwTTP (P < 0.001). HLA- and tumor-related biomarkers had no significant associations with longer rwTTP. As expected, TMB and PD-L1 IHC were significantly associated with longer rwTTP (P < 0.05). In multivariable Cox PH analyses controlling for PD-L1 IHC and TMB, several immune signatures remained significantly associated with rwTTP (table 1). Notably, many RNA-based signatures were highly correlated despite non-overlapping genes and functions, underscoring a broad profile of immune infiltration and activation is likely associated with ICI response.ConclusionsHere we demonstrate that RNA-based immune signatures are significantly associated with clinical benefit and may supplement well-established ICI biomarkers in therapy selection. These immune RNA-based signatures need to be prospectively validated in future studies.ReferencesWang S, Jia M, He Z, Liu XS. APOBEC3B and APOBEC mutational signature as potential predictive markers for immunotherapy response in non-small cell lung cancer. Oncogene. 2018;37(29):3924–3936.Ayers M, Lunceford J, Nebozhyn M, et al. IFN-γ-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest. 2017;127(8):2930–2940.Auslander N, Zhang G, Lee JS, et al. Robust prediction of response to immune checkpoint blockade therapy in metastatic melanoma published correction appears in Nat Med. 2018 Dec;24(12):1942. Nat Med. 2018;24(10):1545–1549.Lau D, Khare S, Stein MM, et al. Integration of tumor extrinsic and intrinsic features associates with immunotherapy response in non-small cell lung cancer. Nat Commun. 2022;13(1):4053.Huang AC, Orlowski RJ, Xu X, et al. A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma. Nat Med. 2019;25(3):454–461.Cabrita R, Lauss M, Sanna A, et al. Tertiary lymphoid structures improve immunotherapy and survival in melanoma published correction appears in Nature. 2020 Apr;580(7801):E1. Nature. 2020;577(7791):561–565.Andersson A, Larsson L, Stenbeck L, et al. Spatial deconvolution of HER2-positive breast cancer delineates tumor-associated cell type interactions. Nat Commun. 2021;12(1):6012.Abstract 181 Table 1ICI biomarker associations with rwTTP in ICI-treated mNSCLC cohort (n=199). Cox PH regression results are shown for each biomarker as a single predictor (Univariate Cox PH HR, P-value) and as a predictor with TMB and PD-Ll IHC included in a multivariable Cox PH model (Multivariable Cox PH HR, P-value).
Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single-cell RNA sequencing (scRNA-Seq) to identify T cell ...receptors (TCRs) in IAR T cells, we have identified a class of TCRs that share TCRα chains between individuals ("public" chains). We isolated IAR T cells from blood of healthy, new-onset T1D and established T1D donors using multiplexed CD154 enrichment and identified paired TCRαβ sequences from 2767 individual cells. More than a quarter of cells shared TCR junctions between 2 or more cells ("expanded"), and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCRα junctions were most prominent in new-onset T1D. Public TCR sequences were more germline like than expanded unique, or "private," TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCRα junctions were often paired with mismatched TCRβ junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity toward distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCRα junctions and germline-constrained antigen recognition properties. Since these "innate-like" TCRs differ from previously described immunodominant TCRβ chains in autoimmunity, they have implications for fundamental studies of disease mechanisms. Self-reactive restricted TCRα chains and their associated epitopes should be considered in fundamental and translational investigations of TCRs in T1D.
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