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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.)

Receptor activator of NFκB ligand (RANKL) is mainly known for its role in bone metabolism, constituting a target for therapeutic interventions. Increasing evidence suggests that RANKL is also involved in oncogenesis and tumor progression, including a prominent role in host-tumor interaction. Our data suggest that targeting RANKL may reinforce natural killer (NK) cell-mediated antitumor responses in patients affected by hematological malignancies.

Natural killer (NK) cells are cytotoxic lymphocytes that largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in acute myeloid leukemia (AML) and application of Rituximab in chronic lymphocytic leukemia (CLL). The tumor necrosis factor (TNF) family member GITR ligand (GITRL) is frequently expressed on leukemia cells in AML and CLL and impairs the reactivity of NK cells which express GITR and upregulate its expression following activation. We developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of GITR-GITRL interaction with targeting leukemia cells for NK antibody-dependent cellular cytotoxicity (ADCC) using GITR-Ig fusion proteins with modified Fc moieties. Neutralization of leukemia-expressed GITRL by the GITR domain enhanced cytotoxicity and cytokine production of NK cells depending on activation state with NK reactivity being further largely dependent on the engineered affinity of the fusion proteins to the Fc receptor. Compared with wild-type GITR-Ig, treatment of primary AML and CLL cells with mutants containing a S239D/I332E modification potently increased cytotoxicity, degranulation, and cytokine production of NK cells in a target-antigen-dependent manner with additive effects being observed with CLL cells upon parallel exposure to Rituximab. Fc-optimized GITR-Ig may thus constitute an attractive means for immunotherapy of leukemia that warrants clinical evaluation.

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.

Bone destruction is a prominent feature of multiple myeloma, but conflicting data exist on the expression and pathophysiologic involvement of the bone remodeling ligand RANKL in this disease and the potential therapeutic benefits of its targeted inhibition. Here, we show that RANKL is expressed by primary multiple myeloma and chronic lymphocytic leukemia (CLL) cells, whereas release of soluble RANKL was observed exclusively with multiple myeloma cells and was strongly influenced by posttranscriptional/posttranslational regulation. Signaling via RANKL into multiple myeloma and CLL cells induced release of cytokines involved in disease pathophysiology. Both the effects of RANKL on osteoclastogenesis and cytokine production by malignant cells could be blocked by disruption of RANK-RANKL interaction with denosumab. As we aimed to combine neutralization of RANKL with induction of antibody-dependent cellular cytotoxicity of natural killer (NK) cells against RANKL-expressing malignant cells and as denosumab does not stimulate NK reactivity, we generated RANK-Fc fusion proteins with modified Fc moieties. The latter displayed similar capacity compared with denosumab to neutralize the effects of RANKL on osteoclastogenesis in vitro, but also potently stimulated NK cell reactivity against primary RANKL-expressing malignant B cells, which was dependent on their engineered affinity to CD16. Our findings introduce Fc-optimized RANK-Ig fusion proteins as attractive tools to neutralize the detrimental function of RANKL while at the same time potently stimulating NK cell antitumor immunity.

Objectives: The receptor activator of the NF-kB ligand (RANKL) pathway is a key mediator of prostate cancer (PC)-induced bone disease. However, little is known about this pathway in patients with non-metastatic PC. We aimed to investigate whether changes of RANKL, its inhibitor osteoprotegerin (OPG) and bone marrow-mesenchymal stromal cells (BM-MSCs) occur in PC patients without manifest bone metastases., Patients and Methods: We determined OPG and soluble RANKL (sRANKL) in serum and corresponding bone marrow (BM) samples of 140 patients before radical prostatectomy by enzyme-linked immunosorbent assay (ELISA). As control serum samples of 50 patients with benign prostate hyperplasia were analyzed. BM mononuclear cells (BMNCs) of 16 PC patients were analyzed for expression of RANKL and CD271 (as marker for MSCs) by flow cytometry., Results: PC patients had significantly lower serum levels of OPG compared to BPH patients (P = 0.007), whereas no differences were observed for serum sRANKL (P = 0.74). Both OPG and sRANKL concentrations of serum and corresponding BM samples correlated significantly (P < 0.0001 each). Interestingly, in PC patients, lower serum and BM OPG levels were associated with a higher proportion of BM-MSCs (P = 0.04 and 0.0016, respectively). No correlations were observed for sRANKL, OPG, BM-MSCs, and established risk parameters of PC., Discussion: The results of the study indicate that localized PC is associated with early specific changes of the RANKL pathway in serum and bone marrow (BM). These changes might be part of the pre-metastatic niche of PC and implicate a potential benefit of RANKL inhibition in patients with localized PC., (Copyright © 2012 Wiley Periodicals, Inc.)

Natural killer (NK) cells play an important role in the immunosurveillance of hematopoietic malignancies. Their reactivity is influenced by activating and inhibitory signals mediated by tumor-expressed ligands for NK receptors. Many members of the tumor necrosis factor (TNF) family modulate differentiation, proliferation, activation and death of both tumor and immune effector cells. The TNF receptor family member glucocorticoid-induced TNFR-related protein (GITR) stimulates anti-tumor immunity in mice, but available data indicate that GITR may mediate different effects in mice and men and impairs the reactivity of human NK cells. Here, we comprehensively studied the expression and function of GITR ligand (GITRL) in leukemia. Among the different leukemia entities, pronounced expression of GITRL on leukemic cells was observed in chronic lymphocytic leukemia (CLL), and the GITR receptor was expressed at significantly higher levels on NK cells of CLL patients compared with healthy controls. Upon GITR-GITRL interaction, signaling via GITRL into the leukemia cells induced the release of interleukin (IL)-6, IL-8 and TNF, which act as growth and survival factors for CLL cells. In addition, GITRL impaired both direct and Rituximab-induced degranulation, cytotoxicity and interferon-γ production of NK cells, which could be restored by GITR blocking antibodies. Thus, GITRL may contribute to disease pathophysiology and resistance to direct and Rituximab-induced NK reactivity in CLL.

NK cells play an important role in tumor immunosurveillance and largely contribute to the therapeutic success of anti-tumor antibodies like Rituximab. Here, we studied the role of the TNF family member 4-1BB ligand (4-1BBL) during the interaction of NK cells with chronic lymphocytic leukemia (CLL) cells. 4-1BBL was highly expressed on patient B-CLL cells in all 56 investigated cases. Signaling via 4-1BBL following interaction with 4-1BB, which was detected on NK cells of CLL patients but not healthy individuals, led to the release of immunoregulatory cytokines including TNF by CLL cells. CLL patient sera contained elevated levels of TNF and induced 4-1BB upregulation on NK cells, which in turn impaired direct and Rituximab-induced NK-cell reactivity against 4-1BBL-expressing targets. NK-cell reactivity was not only enhanced by blocking the interaction of NK cell-expressed 4-1BB with 4-1BBL expressed by CLL cells, but also by preventing 4-1BB upregulation on NK cells via neutralization of TNF in patient serum with Infliximab. Our data indicate that 4-1BBL mediates NK-cell immunosubversion in CLL, and thus might contribute to the reportedly compromised efficacy of Rituximab to induce NK-cell reactivity in the disease, and that TNF neutralization may serve to enhance the efficacy of Rituximab treatment in CLL., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cancer/testis antigens (CTA) are a heterogeneous group of antigens that are expressed preferentially in tumor cells and testis. Based on this definition the human membrane-associated phospholipase A1 beta (lipase family member I, LIPI) has been identified as CTA. The high homology of LIPI and the membrane-associated phospholipase A1 alpha (lipase family member H, LIPH) suggests that both genes are derived from a common ancestor by gene duplication. In contrast to human LIPI, human LIPH is expressed in several tissues. LIPI sequences have only been identified in mammals. Here, we describe the identification of LIPI in non-mammalian vertebrates. Based on the conserved genomic organization of LIPI and LIPH we identified sequences for both lipases in birds and fishes. In all vertebrates the LIPI locus is neighbored by a member of the RNA binding motif (RBM) family, RBM11. By sequencing of reverse transcriptase-polymerase chain reaction products we determined the sequences of LIPI and LIPH messenger RNA from broilers. We found that the sequence homology between LIPI and LIPH is much higher in non-mammalian species than in mammals. In addition, we found broad expression of LIPI in broilers, resembling the expression profile of LIPH. Our data suggest that LIPI is a CTA only in mammalian species and that the unique sequence features of the mammalian LIPI/RBM11 locus have evolved together with the CTA-like expression pattern of LIPI.

The prognosis for patients with advanced stages of Ewing family tumors (EFT) is very poor. EFT express high levels of phosphatidic acid specific membrane-associated phospholipase A1 beta (lipase I, LIPI). LIPI is a cancer/testis antigen and the high tumor specificity suggests that LIPI might be an attractive target for new diagnostic and/or therapeutic developments. By using reverse transcriptase-polymerase chain reaction (RT-PCR), we observed simultaneous presence of multiple LIPI transcript variants in EFT. We cloned and sequenced these transcript variants from EFT cell lines. Sequence analysis indicated that all transcript variants were derived by alternative splicing. Homology modeling of corresponding protein structures suggested that different transcript variants differ in their regulatory lid domains. In addition, expression of receptors for lysophosphatidic acid (LPA) was analyzed in a panel of EFT cell lines by RT-PCR. We observed that EFT cell lines expressed high levels of LPA receptors. Different LIPI transcript variants present in EFT might be involved in the pathogenesis of EFT by signaling via these LPA receptors.

Azacytidine and decitabine are approved for treatment of acute myeloid leukemias and myelodysplastic syndromes. While clinical responses are attributed to epigenetic effects and induction of apoptosis in malignant cells, these azanucleosides also affect antitumor immune responses. NK cells as components of innate immunity may confine development and progression of cancer. Numerous therapeutic strategies presently aim to reinforce NK reactivity against hematopoietic malignancies. We here comparatively analyzed the effect of the two clinically available azanucleosides and report that NK cytotoxicity and IFN-γ production are significantly impaired by pharmacological concentrations of azacytidine but enhanced by decitabine. This was not due to alterations in the target cells but caused by direct effects on NK cells depending on the chemical modifications by which azanucleosides differ from their physiological analogues. Although azacytidine impaired mRNA synthesis and induced apoptosis in NK cells, decitabine did not per se alter NK cell viability or reactivity but enhanced responsiveness to activating stimuli by inducing transcription of genes involved in NK reactivity. Tantalizingly, these effects were independent of incorporation of the azanucleosides into DNA during cell division. While azacytidine impairs NK antitumor immunity, decitabine augments NK reactivity by yet unidentified mechanisms and may thus serve well in therapeutic strategies combining its effects on malignant cells with its ability to enhance NK functions., (Copyright © 2010 UICC.)

Natural killer (NK) cells play an important role in the immunosurveillance of leukemia. Their reactivity is governed by a balance of activating and inhibitory receptors including various members of the tumor necrosis factor receptor (TNFR) family. Here we report that human NK cells acquire expression of the TNFR family member CD137 upon activation, and NK cells of acute myeloid leukemia (AML) patients display an activated phenotype with substantial CD137 expression. CD137 ligand (CD137L) was detectable on leukemic cells in 35% of 65 investigated AML patients, but not on healthy CD34(+) cells, and expression was associated with monocytic differentiation. Bidirectional signaling following CD137-CD137L interaction induced the release of the immunomodulatory cytokines interleukin-10 and TNF by AML cells and directly diminished granule mobilization, cytotoxicity, and interferon-gamma production of human NK cells, which was restored by blocking CD137. Cocultures of NK cells with CD137L transfectants confirmed that human CD137 inhibits NK-cell reactivity, while activating signals were transduced by its counterpart on NK cells in mice. Our data underline the necessity to study the function of seemingly analog immunoregulatory molecules in mice compared with men and demonstrate that CD137-CD137L interaction enables immune evasion of AML cells by impairing NK-cell tumor surveillance in humans.

The reciprocal interaction of tumor cells with the immune system is influenced by various members of the tumor necrosis factor (TNF)/TNF receptor (TNFR) family, and recently, glucocorticoid-induced TNFR-related protein (GITR) was shown to stimulate antitumor immunity in mice. However, GITR may mediate different effects in mice and men and impairs the reactivity of human natural killer (NK) cells. Here, we studied the role of GITR and its ligand (GITRL) in human acute myeloid leukemia (AML). Surface expression of GITRL was observed on AML cells in six of seven investigated cell lines, and 34 of 60 investigated AML patients whereas healthy CD34(+) cells did not express GITRL. Furthermore, soluble GITRL (sGITRL) was detectable in AML patient sera in 18 of 55 investigated cases. While the presence of GITRL was not restricted to a specific AML subtype, surface expression was significantly associated with monocytic differentiation. Signaling via GITRL into patient AML cells induced the release of TNF and interleukin-10 (IL-10), and this was blocked by the inhibition of mitogen-activated protein kinases extracellular signal-regulated kinase 1/2. Furthermore, triggering GITR by surface-expressed and sGITRL impaired NK cell cytotoxicity and IFN-gamma production in cocultures with leukemia cells, and NK cell reactivity could be restored by blocking GITR and neutralization of sGITRL and IL-10. Thus, whereas a stimulatory role of the GITR-GITRL system in mouse antitumor immunity has been reported, our data show that in humans GITRL expression subverts NK cell immunosurveillance of AML. Our results provide useful information for therapeutic approaches in AML, which, like haploidentical stem cell transplantation, rely on a sufficient NK cell response.

NK cell anti-tumor reactivity is governed by a balance of activating and inhibitory receptors including various TNF receptor (TNFR) family members. Here we report that human tumor cells release a soluble form of the TNF family member Glucocorticoid-Induced TNFR-Related Protein (GITR) ligand (sGITRL), which can be detected in cell culture supernatants. Tumor-derived sGITRL concentration-dependently reduced NK cell cytotoxicity and IFN-gamma production, which could be overcome by neutralization of sGITRL using a GITR-Ig fusion protein. Although sGITRL did not induce apoptosis in NK cells, it diminished nuclear localized RelB, indicating that sGITRL negatively modulates NK cell NF-kappaB activity. Furthermore, we detected substantial levels of sGITRL in sera of patients with various malignancies, but not in healthy controls. Presence of sGITRL-containing patient serum in cocultures with tumor cells significantly reduced NK cell cytotoxicity and IFN-gamma production, which could again be restored by neutralization of sGITRL. The strong correlation of tumor incidence and elevated sGITRL levels indicates that sGITRL is released from cancers in vivo, leading to impaired NK cell immunosurveillance of human tumors. Our data suggest that determination of sGITRL levels might be implemented as a tumor marker in patients, and GITRL neutralization may be used to improve immunotherapeutic strategies relying on NK cell reactivity.

Background: Cancer/testis antigens (CTA) represent a heterogeneous group of antigens expressed nearly exclusively in tumour cells and testis. Recently, we identified phospholipase A1 beta (a CTA also known as lipase member I, LIPI) as a gene with high expression in Ewing family tumours (EFT). In the present paper we analyzed expression of LIPI in a panel of normal tissues and tumour samples., Procedure: The expression of CTA in EFT and normal tissues was analyzed by using DNA microarray datasets. Expression of LIPI in EFT, a panel of other tumour samples, and normal tissues was analyzed by using RT-PCR and quantitative RT-PCR., Results: LIPI was expressed in EFT samples but not in other investigated tumour samples. Expression of LIPI in normal tissues was restricted to testis and thyroid. However, expression in these tissues was low compared with EFT. Interestingly testis as well as thyroid expressed all analyzed EFT-associated transcripts, suggesting that these tissues harbour a small cell population with molecular features of EFT. The sensitivity of the LIPI RT-PCR was similar to the sensitivity of the conventional EWSR1-FLI1 RT-PCR, suggesting that LIPI might be useful as additional diagnostic target structure., Conclusions: The human cancer/testis antigen LIPI is highly expressed in Ewing family tumours and can be easily detected by RT-PCR or quantitative RT-PCR. LIPI might be an interesting target for the development of future diagnostic tools or treatment strategies.