Interferon-γ signaling in eosinophilic esophagitis has implications for epithelial barrier function and programmed cell death.

Objective: Eosinophilic esophagitis (EoE) is a chronic esophageal inflammatory disorder characterized by eosinophil-rich mucosal inflammation and tissue remodeling. Transcriptional profiling of esophageal biopsies has previously revealed upregulation of type I and II interferon (IFN) response genes. We aim to unravel interactions between immune and epithelial cells and examine functional significance in esophageal epithelial cells. Design: We investigated epithelial gene expression from EoE patients using single-cell RNA sequencing and a confirmatory bulk RNA-sequencing experiment of isolated epithelial cells. The functional impact of interferon signaling on epithelial cells was investigated using in vitro organoid models. Results: We observe upregulation of interferon response signature genes (ISGs) in the esophageal epithelium during active EoE compared to other cell types, single-cell data, and pathway analyses, identified upregulation in ISGs in epithelial cells isolated from EoE patients. Using an esophageal organoid and air-liquid interface models, we demonstrate that IFN-γ stimulation triggered disruption of esophageal epithelial differentiation, barrier integrity, and induced apoptosis via caspase upregulation. We show that an increase in cleaved caspase-3 is seen in EoE tissue and identify interferon gamma (IFNG) expression predominantly in a cluster of majority-CD8+ T cells with high expression of CD69 and FOS. Conclusion: These findings offer insight into the interplay between immune and epithelial cells in EoE. Our data illustrate the relevance of several IFN-γ-mediated mechanisms on epithelial function in the esophagus, which have the potential to impact epithelial function during inflammatory conditions.

Vaccine-boosted CAR T crosstalk with host immunity to reject tumors with antigen heterogeneity.

Chimeric antigen receptor (CAR) T cell therapy effectively treats human cancer, but the loss of the antigen recognized by the CAR poses a major obstacle. We found that in vivo vaccine boosting of CAR T cells triggers the engagement of the endogenous immune system to circumvent antigen-negative tumor escape. Vaccine-boosted CAR T promoted dendritic cell (DC) recruitment to tumors, increased tumor antigen uptake by DCs, and elicited the priming of endogenous anti-tumor T cells. This process was accompanied by shifts in CAR T metabolism toward oxidative phosphorylation (OXPHOS) and was critically dependent on CAR-T-derived IFN-γ. Antigen spreading (AS) induced by vaccine-boosted CAR T enabled a proportion of complete responses even when the initial tumor was 50% CAR antigen negative, and heterogeneous tumor control was further enhanced by the genetic amplification of CAR T IFN-γ expression. Thus, CAR-T-cell-derived IFN-γ plays a critical role in promoting AS, and vaccine boosting provides a clinically translatable strategy to drive such responses against solid tumors.

Tissue-specific abundance of interferon-gamma drives regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer.

Local environmental factors influence CD8+ T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8+ T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.

Low protease activity in B cell follicles promotes retention of intact antigens after immunization.

The structural integrity of vaccine antigens is critical to the generation of protective antibody responses, but the impact of protease activity on vaccination in vivo is poorly understood. We characterized protease activity in lymph nodes and found that antigens were rapidly degraded in the subcapsular sinus, paracortex, and interfollicular regions, whereas low protease activity and antigen degradation rates were detected in the vicinity of follicular dendritic cells (FDCs). Correlated with these findings, immunization regimens designed to target antigen to FDCs led to germinal centers dominantly targeting intact antigen, whereas traditional immunizations led to much weaker responses that equally targeted the intact immunogen and antigen breakdown products. Thus, spatially compartmentalized antigen proteolysis affects humoral immunity and can be exploited.

Revealing the heterogeneity of CD4+ T cells through single-cell transcriptomics.

Single-cell RNA sequencing (scRNA-seq) offers the ability to resolve whole transcriptomes of single cells with substantial throughput, and it has revolutionized studies of gene expression. The transcriptional resolution available can uncover fine structures of biologic heterogeneity that are manifest among cell populations. Here, we review the applications of scRNA-seq to profile the phenotypes and clonotypes of CD4+ T cells. First, we describe challenges inherent to scRNA-seq that are important for analysis of CD4+ T cells, as well as the technical solutions that are emerging to address these challenges. We then consider major themes of the application of scRNA-seq to CD4+ T cells, including investigation of CD4+ T-cell heterogeneity in model systems, analysis of populations from the peripheral blood, and the profiling of tissue-resident populations. We place emphasis on capabilities unique to scRNA-seq, such as the ability to obtain paired T-cell receptor and transcriptome information from single T cells and the potential to elucidate interactions between CD4+ T cells and other cells in their environment. Finally, we conclude by considering future areas of technologic advancement and innovation through which scRNA-seq may further shape our understanding of the roles of CD4+ T cells in health and disease.

Identification of Highly Cross-Reactive Mimotopes for a Public T Cell Response in Murine Melanoma.

While immune checkpoint blockade results in durable responses for some patients, many others have not experienced such benefits. These treatments rely upon reinvigorating specific T cell-antigen interactions. However, it is often unknown what antigens are being recognized by T cells or how to potently induce antigen-specific responses in a broadly applicable manner. Here, we characterized the CD8+ T cell response to a murine model of melanoma following combination immunotherapy to determine the basis of tumor recognition. Sequencing of tumor-infiltrating T cells revealed a repertoire of highly homologous TCR sequences that were particularly expanded in treated mice and which recognized an antigen from an endogenous retrovirus. While vaccination against this peptide failed to raise a protective T cell response in vivo, engineered antigen mimotopes induced a significant expansion of CD8+ T cells cross-reactive to the original antigen. Vaccination with mimotopes resulted in killing of antigen-loaded cells in vivo yet showed modest survival benefit in a prophylactic vaccine paradigm. Together, this work demonstrates the identification of a dominant tumor-associated antigen and generation of mimotopes which can induce robust functional T cell responses that are cross-reactive to the endogenous antigen across multiple individuals.

WAT3R: recovery of T-cell receptor variable regions from 3' single-cell RNA-sequencing.

SUMMARY: Diversity of the T-cell receptor (TCR) repertoire is central to adaptive immunity. The TCR is composed of α and ß chains, encoded by the TRA and TRB genes, of which the variable regions determine antigen specificity. To generate novel biological insights into the complex functioning of immune cells, combined capture of variable regions and single-cell transcriptomes provides a compelling approach. Recent developments enable the enrichment of TRA and TRB variable regions from widely used technologies for 3'-based single-cell RNA-sequencing (scRNA-seq). However, a comprehensive computational pipeline to process TCR-enriched data from 3' scRNA-seq is not available. Here, we present an analysis pipeline to process TCR variable regions enriched from 3' scRNA-seq cDNA. The tool reports TRA and TRB nucleotide and amino acid sequences linked to cell barcodes, enabling the reconstruction of T-cell clonotypes with associated transcriptomes. We demonstrate the software using peripheral blood mononuclear cells from a healthy donor and detect TCR sequences in a high proportion of single T cells. Detection of TCR sequences is low in non-T-cell populations, demonstrating specificity. Finally, we show that TCR clones are larger in CD8 Memory T cells than in other T-cell types, indicating an association between T-cell clonotypes and differentiation states. AVAILABILITY AND IMPLEMENTATION: The Workflow for Association of T-cell receptors from 3' single-cell RNA-seq (WAT3R), including test data, is available on GitHub (https://github.com/mainciburu/WAT3R), Docker Hub (https://hub.docker.com/r/mainciburu/wat3r) and a workflow on the Terra platform (https://app.terra.bio). The test dataset is available on GEO (accession number GSE195956). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations.

The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3' single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis.

Peanut oral immunotherapy differentially suppresses clonally distinct subsets of T helper cells.

Food allergy affects an estimated 8% of children in the United States. Oral immunotherapy (OIT) is a recently approved treatment, with outcomes ranging from sustained tolerance to food allergens to no apparent benefit. The immunological underpinnings that influence clinical outcomes of OIT remain largely unresolved. Using single-cell RNA-Seq and paired T cell receptor α/ß (TCRα/ß) sequencing, we assessed the transcriptomes of CD154+ and CD137+ peanut-reactive T helper (Th) cells from 12 patients with peanut allergy longitudinally throughout OIT. We observed expanded populations of cells expressing Th1, Th2, and Th17 signatures that further separated into 6 clonally distinct subsets. Four of these subsets demonstrated a convergence of TCR sequences, suggesting antigen-driven T cell fates. Over the course of OIT, we observed suppression of Th2 and Th1 gene signatures in effector clonotypes but not T follicular helper-like (Tfh-like) clonotypes. Positive outcomes were associated with stronger suppression of Th2 signatures in Th2A-like cells, while treatment failure was associated with the expression of baseline inflammatory gene signatures that were present in Th1 and Th17 cell populations and unmodulated by OIT. These results demonstrate that differential clinical responses to OIT are associated with both preexisting characteristics of peanut-reactive CD4+ T cells and suppression of a subset of Th2 cells.

Lack of CD8+ T cell effector differentiation during priming mediates checkpoint blockade resistance in non-small cell lung cancer.

In non­small cell lung cancer (NSCLC), response to immune checkpoint blockade (ICB) is associated with programmed cell death ligand 1 expression that is induced by interferon-γ­producing, tumor-infiltrating CD8+ T cells. However, not all tumors with a CD8+ T cell infiltrate respond to ICB, and little is known about the mechanisms governing ICB resistance in T cell­infiltrated NSCLC. We used an orthotopic NSCLC mouse model to study ICB-refractory CD8+ T cell responses. Single-cell RNA sequencing of the NSCLC mouse tumors revealed that lung cancer­specific tumor-infiltrating CD8+ T cells exhibited clonal expansion but lacked expression of genes associated with effector and exhausted T cell responses, indicating that they underwent a differentiation program distinct from conventional T cell exhaustion. This lung cancer­specific T cell dysfunction program was established early during priming in the mediastinal lymph node and was characterized by robust proliferation but a failed up-regulation of effector and exhausted T cell characteristics. Intriguingly, CD8+ T cells from patients with NSCLC expressed an analogous gene expression program, which appeared distinct from conventional T cell exhaustion. Administration of recombinant interleukin-2 (IL-2) and IL-12 was sufficient to restore effector T cell differentiation and induce control of KP lung tumors. These findings imply that a CD8+ T cell differentiation trajectory, activated during T cell priming in the mediastinal lymph node, limits the response of CD8+ T cells to ICB and thereby may contribute to failure of ICB in a subset T cell­infiltrated NSCLC.

Clonally expanded, GPR15-expressing pathogenic effector TH2 cells are associated with eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is an allergic disorder characterized by the recruitment of eosinophils to the esophagus, resulting in chronic inflammation. We sought to understand the cellular populations present in tissue biopsies of patients with EoE and to determine how these populations are altered between active disease and remission. To this end, we analyzed cells obtained from esophageal biopsies, duodenal biopsies, and peripheral blood of patients with EoE diagnosed with active disease or remission with single-cell RNA and T cell receptor (TCR) sequencing. Pathogenic effector TH2 (peTH2) cells present in the esophageal biopsies of patients with active disease expressed distinct gene signatures associated with the synthesis of eicosanoids. The esophageal tissue-resident peTH2 population also exhibited clonal expansion, suggesting antigen-specific activation. Peripheral CRTH2+CD161- and CRTH2+CD161+ memory CD4+ T cells were enriched for either a conventional TH2 phenotype or a peTH2 phenotype, respectively. These cells also exhibited substantial clonal expansion and convergence of TCR sequences, suggesting that they are expanded in response to a defined set of antigens. The esophagus-homing receptor GPR15 was up-regulated by peripheral peTH2 clonotypes that were also detected in the esophagus. Finally, GPR15+ peTH2 cells were enriched among milk-reactive CD4+ T cells in patients with milk-triggered disease, suggesting that these cells are an expanded, food antigen-specific population with enhanced esophagus homing potential.

TCR sequencing paired with massively parallel 3' RNA-seq reveals clonotypic T cell signatures.

High-throughput 3' single-cell RNA-sequencing (scRNA-seq) allows cost-effective, detailed characterization of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Here, we present a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 3'-barcoded scRNA-seq samples. This approach is compatible with common 3' scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with T cells sharing common TCRs from immunized mice and from patients with food allergy. We observed preferential phenotypes among subsets of expanded clonotypes, including type 2 helper CD4+ T cell (TH2) states associated with food allergy. These results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.