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Brain tumors in children are a devastating disease in a high proportion of patients. Owing to inconsistent results in clinical trials in unstratified patients, the role of immunotherapy remains unclear. We performed an in-depth survey of the single-cell transcriptomes and clonal relationship of intra-tumoral T cells from children with brain tumors. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with the potential to recognize tumor antigens. Such clonally expanded T cells display enrichment of transcripts linked to effector function, tissue residency, immune checkpoints and signatures of neoantigen-specific T cells and immunotherapy response. We identify neoantigens in pediatric brain tumors and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes. Notably, among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and magnitude of T cell response. Our findings suggest that characterization of intra-tumoral T cell responses may enable selection of patients for immunotherapy, an approach that requires prospective validation in clinical trials., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

With the continued promise of immunotherapy for treating cancer, understanding how host genetics contributes to the tumor immune microenvironment (TIME) is essential to tailoring cancer screening and treatment strategies. Here, we study 1084 eQTLs affecting the TIME found through analysis of The Cancer Genome Atlas and literature curation. These TIME eQTLs are enriched in areas of active transcription, and associate with gene expression in specific immune cell subsets, such as macrophages and dendritic cells. Polygenic score models built with TIME eQTLs reproducibly stratify cancer risk, survival and immune checkpoint blockade (ICB) response across independent cohorts. To assess whether an eQTL-informed approach could reveal potential cancer immunotherapy targets, we inhibit CTSS, a gene implicated by cancer risk and ICB response-associated polygenic models; CTSS inhibition results in slowed tumor growth and extended survival in vivo. These results validate the potential of integrating germline variation and TIME characteristics for uncovering potential targets for immunotherapy., (© 2023. The Author(s).)

The impact of genetic variants on cells challenged in biologically relevant contexts has not been fully explored. Here, we activated CD4 + T cells from 89 healthy donors and performed a single-cell RNA sequencing assay with >1 million cells to examine cell type-specific and activation-dependent effects of genetic variants. Single-cell expression quantitative trait loci (sc-eQTL) analysis of 19 distinct CD4 + T cell subsets showed that the expression of over 4000 genes is significantly associated with common genetic polymorphisms and that most of these genes show their most prominent effects in specific cell types. These genes included many that encode for molecules important for activation, differentiation, and effector functions of T cells. We also found new gene associations for disease-risk variants identified from genome-wide association studies and highlighted the cell types in which their effects are most prominent. We found that biological sex has a major influence on activation-dependent gene expression in CD4 + T cell subsets. Sex-biased transcripts were significantly enriched in several pathways that are essential for the initiation and execution of effector functions by CD4 + T cells like TCR signaling, cytokines, cytokine receptors, costimulatory, apoptosis, and cell-cell adhesion pathways. Overall, this DICE (Database of Immune Cell Expression, eQTLs, and Epigenomics) subproject highlights the power of sc-eQTL studies for simultaneously exploring the activation and cell type-dependent effects of common genetic variants on gene expression (https://dice-database.org).

Common genetic polymorphisms associated with COVID-19 illness can be utilized for discovering molecular pathways and cell types driving disease pathogenesis. Given the importance of immune cells in the pathogenesis of COVID-19 illness, here we assessed the effects of COVID-19-risk variants on gene expression in a wide range of immune cell types. Transcriptome-wide association study and colocalization analysis revealed putative causal genes and the specific immune cell types where gene expression is most influenced by COVID-19-risk variants. Notable examples include OAS1 in non-classical monocytes, DTX1 in B cells, IL10RB in NK cells, CXCR6 in follicular helper T cells, CCR9 in regulatory T cells and ARL17A in T H 2 cells. By analysis of transposase accessible chromatin and H3K27ac-based chromatin-interaction maps of immune cell types, we prioritized potentially functional COVID-19-risk variants. Our study highlights the potential of COVID-19 genetic risk variants to impact the function of diverse immune cell types and influence severe disease manifestations., (© 2021. The Author(s).)

Systemic lupus erythematosus (SLE) is an incurable autoimmune disease disproportionately affecting women. A major obstacle in finding targeted therapies for SLE is its remarkable heterogeneity in clinical manifestations as well as in the involvement of distinct cell types. To identify cell-specific targets as well as cross-correlation relationships among expression programs of different cell types, we here analyze six major circulating immune cell types from SLE patient blood. Our results show that presence of an interferon response signature stratifies patients into two distinct groups (IFNneg vs. IFNpos). Comparing these two groups using differential gene expression and differential gene coexpression analysis, we prioritize a relatively small list of genes from classical monocytes including two known immune modulators: TNFSF13B/BAFF (target of belimumab , an approved therapeutic for SLE) and IL1RN (the basis of anakinra, a therapeutic for rheumatoid arthritis). We then develop a multi-cell type extension of the weighted gene coexpression network analysis (WGCNA) framework, termed mWGCNA. Applying mWGCNA to RNA-seq data from six sorted immune cell populations (15 SLE, 10 healthy donors), we identify a coexpression module with interferon-stimulated genes (ISGs) among all cell types and a cross-cell type correlation linking expression of specific T helper cell markers to B cell response as well as to TNFSF13B expression from myeloid cells, all of which in turn correlates with disease severity of IFNpos patients. Our results demonstrate the power of a hypothesis-free and data-driven approach to discover drug targets and to reveal novel cross-correlation across cell types in SLE with implications for other autoimmune diseases., (© 2021 Panwar et al.; Published by Cold Spring Harbor Laboratory Press.)

Expression quantitative trait loci (eQTLs) studies provide associations of genetic variants with gene expression but fall short of pinpointing functionally important eQTLs. Here, using H3K27ac HiChIP assays, we mapped eQTLs overlapping active cis-regulatory elements that interact with their target gene promoters (promoter-interacting eQTLs, pieQTLs) in five common immune cell types (Database of Immune Cell Expression, Expression quantitative trait loci and Epigenomics (DICE) cis-interactome project). This approach allowed us to identify functionally important eQTLs and show mechanisms that explain their cell-type restriction. We also devised an approach to eQTL discovery that relies on HiChIP-based promoter interaction maps as a structural framework for deciding which SNPs to test for association with gene expression, and observe ultra-long-distance pieQTLs (>1 megabase away), including several disease-risk variants. We validated the functional role of pieQTLs using reporter assays, CRISPRi, dCas9-tiling guides and Cas9-mediated base-pair editing. In this article we present a method for functional eQTL discovery and provide insights into relevance of noncoding variants for cell-specific gene regulation and for disease association beyond conventional eQTL mapping.

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (>100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

CD4 + T helper (T H ) cells and regulatory T (T reg ) cells that respond to common allergens play an important role in driving and dampening airway inflammation in patients with asthma. Until recently, direct, unbiased molecular analysis of allergen-reactive T H and T reg cells has not been possible. To better understand the diversity of these T cell subsets in allergy and asthma, we analyzed the single-cell transcriptome of ~50,000 house dust mite (HDM) allergen-reactive T H cells and T reg cells from asthmatics with HDM allergy and from three control groups: asthmatics without HDM allergy and nonasthmatics with and without HDM allergy. Our analyses show that HDM allergen-reactive T H and T reg cells are highly heterogeneous and certain subsets are quantitatively and qualitatively different in individuals with HDM-reactive asthma. The number of interleukin-9 (IL-9)-expressing HDM-reactive T H cells is greater in asthmatics with HDM allergy compared with nonasthmatics with HDM allergy, and this IL-9-expressing T H subset displays enhanced pathogenic properties. More HDM-reactive T H and T reg cells expressing the interferon response signature (T H IFNR and T reg IFNR) are present in asthmatics without HDM allergy compared with those with HDM allergy. In cells from these subsets (T H IFNR and T reg IFNR), expression of TNFSF10 was enriched; its product, tumor necrosis factor-related apoptosis-inducing ligand, dampens activation of T H cells. These findings suggest that the T H IFNR and T reg IFNR subsets may dampen allergic responses, which may help explain why only some people develop T H 2 responses to nearly ubiquitous allergens., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Genetic variants at the PTPN2 locus, which encodes the tyrosine phosphatase PTPN2, cause reduced gene expression and are linked to rheumatoid arthritis (RA) and other autoimmune diseases. PTPN2 inhibits signaling through the T cell and cytokine receptors, and loss of PTPN2 promotes T cell expansion and CD4- and CD8-driven autoimmunity. However, it remains unknown whether loss of PTPN2 in FoxP3+ regulatory T cells (Tregs) plays a role in autoimmunity. Here we aimed to model human autoimmune-predisposing PTPN2 variants, the presence of which results in a partial loss of PTPN2 expression, in mouse models of RA. We identified that reduced expression of Ptpn2 enhanced the severity of autoimmune arthritis in the T cell-dependent SKG mouse model and demonstrated that this phenotype was mediated through a Treg-intrinsic mechanism. Mechanistically, we found that through dephosphorylation of STAT3, PTPN2 inhibits IL-6-driven pathogenic loss of FoxP3 after Tregs have acquired RORγt expression, at a stage when chromatin accessibility for STAT3-targeted IL-17-associated transcription factors is maximized. We conclude that PTPN2 promotes FoxP3 stability in mouse RORγt+ Tregs and that loss of function of PTPN2 in Tregs contributes to the association between PTPN2 and autoimmunity.

While many genetic variants have been associated with risk for human diseases, how these variants affect gene expression in various cell types remains largely unknown. To address this gap, the DICE (database of immune cell expression, expression quantitative trait loci [eQTLs], and epigenomics) project was established. Considering all human immune cell types and conditions studied, we identified cis-eQTLs for a total of 12,254 unique genes, which represent 61% of all protein-coding genes expressed in these cell types. Strikingly, a large fraction (41%) of these genes showed a strong cis-association with genotype only in a single cell type. We also found that biological sex is associated with major differences in immune cell gene expression in a highly cell-specific manner. These datasets will help reveal the effects of disease risk-associated genetic polymorphisms on specific immune cell types, providing mechanistic insights into how they might influence pathogenesis (https://dice-database.org)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The TNF receptor family member OX40 promotes activation and proliferation of T cells, which fuels efforts to modulate this immune checkpoint to reinforce antitumor immunity. Besides T cells, NK cells are a second cytotoxic lymphocyte subset that contributes to antitumor immunity, particularly in leukemia. Accordingly, these cells are being clinically evaluated for cancer treatment through multiple approaches, such as adoptive transfer of ex vivo expanded polyclonal NK cells (pNKC). Here, we analyzed whether and how OX40 and its ligand (OX40L) influence NK-cell function and antileukemia reactivity. We report that OX40 is expressed on leukemic blasts in a substantial percentage of patients with acute myeloid leukemia (AML) and that OX40 can, after stimulation with agonistic OX40 antibodies, mediate proliferation and release of cytokines that act as growth and survival factors for the leukemic cells. We also demonstrate that pNKC differentially express OX40L, depending on the protocol used for their generation. OX40L signaling promoted NK-cell activation, cytokine production, and cytotoxicity, and disruption of OX40-OX40L interaction impaired pNKC reactivity against primary AML cells. Together, our data implicate OX40/OX40L in disease pathophysiology of AML and in NK-cell immunosurveillance. Our findings indicate that effects of the OX40-OX40L receptor-ligand system in other immune cell subsets and also malignant cells should be taken into account when developing OX40-targeted approaches for cancer immunotherapy. Cancer Immunol Res; 6(2); 209-21. ©2018 AACR ., (©2018 American Association for Cancer Research.)

CD4 + cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, and heterogeneity, especially in relation to other well-described CD4 + memory T cell subsets. We performed single-cell RNA sequencing in more than 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile, and clonality in humans. Single-cell differential gene expression analysis revealed a spectrum of known transcripts, including several linked to cytotoxic and costimulatory function that are expressed at higher levels in the T EMRA (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4 + T cells in the central memory (T CM ) and effector memory (T EM ) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-T EMRA cells show marked clonal expansion compared with T CM and T EM cells and that most of CD4-T EMRA were dengue virus (DENV)-specific in donors with previous DENV infection. The profile of CD4-T EMRA was highly heterogeneous across donors, with four distinct clusters identified by the single-cell analysis. We identified distinct clusters of CD4-CTL effector and precursor cells in the T EMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and, thus, could provide insights into the mechanisms that may be used to generate durable and effective CD4-CTL immunity., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Transcriptomic profiling by RNA sequencing (RNA-Seq) represents the preferred approach to measure genome-wide gene expression for understanding cellular function, tissue development, disease pathogenesis, as well as to identify potential biomarkers and therapeutic targets. For samples with small cell numbers, multiple methods have been described to increase the efficiency of library preparation and to reduce hands-on time and costs. This chapter reviews our approach, which combines flow cytometry and the most recent high-resolution techniques to perform RNA-Seq for samples with low cell numbers as well as for single-cell samples. Our approach reduces technical variability while increasing sensitivity and efficiency. Thus, it is well-suited for large-scale gene expression profiling studies with limited samples for basic and clinical studies.

The Chapter was inadvertently published without Acknowledgement. We have now added the acknowledgement in the chapter. Please find the same below.

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is the preferred approach to map histone modifications and identify cis-regulatory DNA elements throughout the genome. Multiple methods have been described to increase the efficiency of library preparation and to reduce hands-on time as well as costs. This review describes detailed steps to perform cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation for a batch of 48-96 samples with small cell numbers. The protocol implements a semiautomated platform to reduce technical variability and improve signal-to-noise ratio as well as reduce hands-on time, thus allowing large-scale epigenetic studies of clinical samples with limited cell numbers.

Asthma and autoimmune disease susceptibility has been strongly linked to genetic variants in the 17q21 haploblock that alter the expression of ORMDL3; however, the molecular mechanisms by which these variants perturb gene expression and the cell types in which this effect is most prominent are unclear. We found several 17q21 variants overlapped enhancers present mainly in primary immune cell types. CD4 + T cells showed the greatest increase (threefold) in ORMDL3 expression in individuals carrying the asthma-risk alleles, where ORMDL3 negatively regulated interleukin-2 production. The asthma-risk variants rs4065275 and rs12936231 switched CTCF-binding sites in the 17q21 locus, and 4C-Seq assays showed that several distal cis-regulatory elements upstream of the disrupted ZPBP2 CTCF-binding site interacted with the ORMDL3 promoter region in CD4 + T cells exclusively from subjects carrying asthma-risk alleles. Overall, our results suggested that T cells are one of the most prominent cell types affected by 17q21 variants.

The epidermal growth factor receptor HER2/neu is expressed on various cancers and represents a negative prognostic marker, but is also a target for the therapeutic monoclonal antibody Trastuzumab. In about 30% of cases, HER2/neu is expressed on acute lymphoblastic leukemia (ALL) cells and was proposed to be associated with a deleterious prognosis. Here we evaluated clinical data from 65 ALL patients (HER2/neu+, n = 17; HER2/neu-, n = 48) with a median follow-up of 19.4 months (range 0.6-176.5 months) and observed no association of HER2/neu expression with response to chemotherapy, disease free or overall survival. In vitro, treatment of primary ALL cells (CD20+HER2/neu+, CD20+HER2/neu- and CD20-HER2/neu-) with Rituximab and Trastuzumab led to activation of NK cells in strict dependence of the expression of the respective antigen. NK reactivity was more pronounced with Rituximab as compared to Trastuzumab, and combined application could lead to additive effects in cases where both antigens were expressed. Besides providing evidence that HER2/neu expression is no risk factor in ALL patients, our data demonstrates that HER2/neu can be a promising target for Trastuzumab therapy in the subset of ALL patients with the potential to improve disease outcome.

Natural killer (NK) cells are cytotoxic lymphocytes that substantially contribute to the therapeutic benefit of antitumor antibodies like Rituximab, a crucial component in the treatment of B-cell malignancies. In chronic lymphocytic leukemia (CLL), the ability of NK cells to lyse the malignant cells and to mediate antibody-dependent cellular cytotoxicity upon Fc receptor stimulation is compromised, but the underlying mechanisms are largely unclear. We report here that NK-cells activation-dependently produce the tumor necrosis factor family member 'B-cell activating factor' (BAFF) in soluble form with no detectable surface expression, also in response to Fc receptor triggering by therapeutic CD20-antibodies. BAFF in turn enhanced the metabolic activity of primary CLL cells and impaired direct and Rituximab-induced lysis of CLL cells without affecting NK reactivity per se. The neutralizing BAFF antibody Belimumab, which is approved for treatment of systemic lupus erythematosus, prevented the effects of BAFF on the metabolism of CLL cells and restored their susceptibility to direct and Rituximab-induced NK-cell killing in allogeneic and autologous experimental systems. Our findings unravel the involvement of BAFF in the resistance of CLL cells to NK-cell antitumor immunity and Rituximab treatment and point to a benefit of combinatory approaches employing BAFF-neutralizing drugs in B-cell malignancies.

Recruitment of Fc-receptor-bearing effector cells, such as natural killer (NK) cells, is a feature critical for the therapeutic success of antitumor antibodies and can be improved by the modifications of an antibody's Fc part. The various ligands of the activating immunoreceptor NKG2D, NKG2DL) are selectively expressed on malignant cells including leukemia. We here took advantage of the tumor-associated expression of NKG2DL for targeting leukemic cells by NKG2D-immunoglobulin G (IgG)1 fusion proteins containing modified Fc parts. Compared to NKG2D-Fc containing a wild-type Fc part (NKG2D-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/ΔG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell Fc receptor, respectively. Functional analyses with allogenic as well as autologous NK cells and primary malignant cells of leukemia patients revealed that NKG2D-Fc-KO significantly reduced NK reactivity by blocking immunostimulatory NKG2D-NKG2DL interaction. NKG2D-Fc-WT already enhanced antileukemia reactivity by inducing antibody-dependent cellular cytotoxicity (ADCC) with NKG2D-Fc-ADCC mediating significantly stronger effects. Parallel application of NKG2D-Fc-ADCC with Rituximab caused additive effects in lymphoid leukemia. In line with the tumor-associated expression of NKG2DL, no NK cell ADCC against resting healthy blood cells was induced. Thus, NKG2D-Fc-ADCC potently enhances NK antileukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL and may constitute an attractive means for immunotherapy of leukemia., (© 2014 UICC.)

The ability of NK cells to mediate Ab-dependent cellular cytotoxicity (ADCC) largely contributes to the clinical success of antitumor Abs, including trastuzumab, which is approved for the treatment of breast cancer with HER2/neu overexpression. Notably, only ∼25% of breast cancer patients overexpress HER2/neu. Moreover, HER2/neu is expressed on healthy cells, and trastuzumab application is associated with side effects. In contrast, the ligands of the activating immunoreceptor NKG2D (NKG2DL) are selectively expressed on malignant cells. In this study, we took advantage of the tumor-associated expression of NKG2DL by using them as target Ags for NKG2D-IgG1 fusion proteins optimized by amino acid exchange S239D/I332E in their Fc part. Compared to constructs with wild-type Fc parts, fusion proteins carrying the S239D/I332E modification (NKG2D-Fc-ADCC) mediated highly enhanced degranulation, ADCC, and IFN-γ production of NK cells in response to breast cancer cells. NKG2D-Fc-ADCC substantially enhanced NK reactivity also against HER2/neu-low targets that were unaffected by trastuzumab, as both compounds mediated their immunostimulatory effects in strict dependence of target Ag expression levels. Thus, in line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to its highly increased affinity to CD16, NKG2D-Fc-ADCC potently enhances NK cell reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, NKG2D-Fc-ADCC may constitute an attractive means for immunotherapy especially of HER2/neu-low or -negative breast cancer., (Copyright © 2014 by The American Association of Immunologists, Inc.)