Single-cell transcriptome analysis reveals coordinated ectopic gene-expression patterns in medullary thymic epithelial cells.

Expression of tissue-restricted self antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for the induction of self-tolerance and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and is coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA sequencing and obtained evidence of numerous recurring TRA-co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process that might involve local remodeling of chromatin and thus ensures a comprehensive representation of the immunological self.

Reimagining antibody-dependent cellular cytotoxicity in cancer: the potential of natural killer cell engagers.

Bi-, tri- and multispecific antibodies have enabled the development of targeted cancer immunotherapies redirecting immune effector cells to eliminate malignantly transformed cells. These antibodies allow for simultaneous binding of surface antigens on malignant cells and activating receptors on innate immune cells, such as natural killer (NK) cells, macrophages, and neutrophils. Significant progress with such antibodies has been achieved, particularly in hematological malignancies. Nevertheless, several major challenges remain, including increasing their immunotherapeutic efficacy in a greater proportion of patients, particularly in those harboring solid tumors, and overcoming dose-limiting toxicities and immunogenicity. Here, we discuss novel antibody-engineering developments designed to maximize the potential of NK cells by NK cell engagers mediating antibody-dependent cellular cytotoxicity (ADCC), thereby expanding the armamentarium for cancer immunotherapy.

Thymic B Cells Are Licensed to Present Self Antigens for Central T Cell Tolerance Induction.

Thymic antigen-presenting cells (APCs) such as dendritic cells and medullary thymic epithelial cells (mTECs) use distinct strategies of self-antigen expression and presentation to mediate central tolerance. The thymus also harbors B cells; whether they also display unique tolerogenic features and how they genealogically relate to peripheral B cells is unclear. Here, we found that Aire is expressed in thymic but not peripheral B cells. Aire expression in thymic B cells coincided with major histocompatibility class II (MHCII) and CD80 upregulation and immunoglobulin class-switching. These features were recapitulated upon immigration of naive peripheral B cells into the thymus, whereby this intrathymic licensing required CD40 signaling in the context of cognate interactions with autoreactive CD4(+) thymocytes. Moreover, a licensing-dependent neo-antigen selectively upregulated in immigrating B cells mediated negative selection through direct presentation. Thus, autoreactivity within the nascent T cell repertoire fuels a feed forward loop that endows thymic B cells with tolerogenic features.

Discovery and optimization of substituted oxalamides as novel heme-displacing IDO1 inhibitors.

Since the advent of antibody checkpoint inhibitors as highly efficient drugs for cancer treatment, the development of immunomodulating small molecules in oncology has gained great attention. Drug candidates targeting IDO1, a key enzyme in tryptophan metabolism, are currently under clinical investigation in combination with PD-1/PD-L1 agents as well as with other established anti-tumor therapeutics. A ligand based design approach from hydroxyamidine 4 that aimed at heme-binding IDO1 inhibitors resulted in new compounds with moderate IDO1 potency. A hybrid structure design that made use of the linrodostat structure (2) led to oxalamide derived, heme-displacing IDO1 inhibitors with high cell-based IDO1 potency and a favorable ADME/PK profile.

Discovery of highly potent heme-displacing IDO1 inhibitors based on a spirofused bicyclic scaffold.

Through structural modification of an oxalamide derived chemotype, a novel class of highly potent, orally bioavailable IDO1-specific inhibitors was identified. Representative compound 18 inhibited human IDO1 with IC50 values of 3.9 nM and 52 nM in a cellular and human whole blood assay, respectively. In vitro assessment of the ADME properties of 18 demonstrated very high metabolic stability. Pharmacokinetic profiling in mice showed a significantly reduced clearance compared to the oxalamides. In a mouse pharmacodynamic model 18 nearly completely suppressed lipopolysaccharide-induced kynurenine production. Hepatocyte data of 18 suggest the human clearance to be in a similar range to linrodostat (1).

Revisiting the Road Map of Medullary Thymic Epithelial Cell Differentiation.

The basic two-step terminal differentiation model of the medullary thymic epithelial cell (mTEC) lineage from immature MHC class II (MHCII)lo to mature MHCIIhi mTECs has recently been extended to include a third stage, namely the post-Aire MHCIIlo subset as identified by lineage-tracing models. However, a suitable surface marker distinguishing the phenotypically overlapping pre- from the post-Aire MHCIIlo stage has been lacking. In this study, we introduce the lectin Tetragonolobus purpureas agglutinin (TPA) as a novel cell surface marker that allows for such delineation. Based on our data, we derived the following sequence of mTEC differentiation: TPAloMHCIIlo → TPAloMHCIIhi → TPAhiMHCIIhi → TPAhiMHCIIlo Surprisingly, in the steady-state postnatal thymus TPAloMHCIIlo pre-Aire rather than terminally differentiated post-Aire TPAhiMHCIIlo mTECs were marked for apoptosis at an exceptionally high rate of ∼70%. Hence, only the minor cycling fraction of the MHCIIlo subset (<20%) potentially qualified as mTEC precursors. FoxN1 expression inversely correlated with the fraction of slow cycling and apoptotic cells within the four TPA subsets. TPA also further subdivided human mTECs, although with different subset distribution. Our revised road map emphazises close parallels of terminal mTEC development with that of skin, undergoing an alternative route of cell death, namely cornification rather than apoptosis. The high rate of apoptosis in pre-Aire MHCIIlo mTECs points to a "quality control" step during early mTEC differentiation.

Dissecting and modeling the emergent murine TEC compartment during ontogeny.

The origin of the thymic epithelium, i.e. the cortical (cTEC) and medullary (mTEC) epithelial cells, from bipotent stem cells through TEC progenitors and lineage-specific progeny still remains poorly understood. We sought to obtain an unbiased view of the incipient emergence of TEC subsets by following embryonic TEC development based on co-expression of EpCAM, CD80 and MHC class II (MHCII) on non-hematopoietic (CD45- ) thymic stromal cells in wild-type BL6 mice. Using a combination of ex vivo analysis, Re-aggregate Thymic Organ Culture (RTOC) reconstitution assays and mathematical modeling, we traced emergent lineage commitment in murine embryonic TECs. Both experimental and mathematical datasets supported the following developmental sequence: MHCII- CD80- → MHCIIlo CD80- → MHCIIhi CD80- → MHCIIhi CD80hi TECs, whereby MHCIIhi CD80- and MHCIIhi CD80hi TECs bear features of cTECs and mTECs respectively. These emergent MHCIIhi CD80- cTECs directly generate mature MHCIIhi CD80hi mTECs in vivo and in vitro, thus supporting the asynchronous model of TEC lineage commitment.

Overlapping gene coexpression patterns in human medullary thymic epithelial cells generate self-antigen diversity.

Promiscuous expression of numerous tissue-restricted self-antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential to safeguard self-tolerance. A distinct feature of promiscuous gene expression is its mosaic pattern (i.e., at a given time, each self-antigen is expressed only in 1-3% of mTECs). How this mosaic pattern is generated at the single-cell level is currently not understood. Here, we show that subsets of human mTECs expressing a particular TRA coexpress distinct sets of genes. We identified three coexpression groups comprising overlapping and complementary gene sets, which preferentially mapped to certain chromosomes and intrachromosomal gene clusters. Coexpressed gene loci tended to colocalize to the same nuclear subdomain. The TRA subsets aligned along progressive differentiation stages within the mature mTEC subset and, in vitro, interconverted along this sequence. Our data suggest that single mTECs shift through distinct gene pools, thus scanning a sizeable fraction of the overall repertoire of promiscuously expressed self-antigens. These findings have implications for the temporal and spatial (re)presentation of self-antigens in the medulla in the context of tolerance induction.

Misinitiation of intrathymic MART-1 transcription and biased TCR usage explain the high frequency of MART-1-specific T cells.

Immunity to tumor differentiation antigens, such as melanoma antigen recognized by T cells 1 (MART-1), has been comprehensively studied. Intriguingly, CD8(+) T cells specific for the MART-1(26(27)-35) epitope in the context of HLA-A0201 are about 100 times more abundant compared with T cells specific for other tumor-associated antigens. Moreover, MART-1-specific CD8(+) T cells show a highly biased usage of the Vα-region gene TRAV12-2. Here, we provide independent support for this notion, by showing that the combinatorial pairing of different TCRα- and TCRß- chains derived from HLA-A2-MART-1(26-35) -specific CD8(+) T-cell clones is unusually permissive in conferring MART-1 specificity, provided the CDR1α TRAV12-2 region is used. Whether TCR bias alone accounts for the unusual abundance of HLA-A2-MART-1(26-35) -specific CD8(+) T cells has remained conjectural. Here, we provide an alternative explanation: misinitiated transcription of the MART-1 gene resulting in truncated mRNA isoforms leads to lack of promiscuous transcription of the MART-1(26-35) epitope in human medullary thymic epithelial cells and, consequently, evasion of central self-tolerance toward this epitope. Thus, biased TCR usage and leaky central tolerance might act in an independent and additive manner to confer high frequency of MART-1(26-35) -specific CD8(+) T cells.

An organotypic coculture model supporting proliferation and differentiation of medullary thymic epithelial cells and promiscuous gene expression.

Understanding intrathymic T cell differentiation has been greatly aided by the development of various reductionist in vitro models that mimic certain steps/microenvironments of this complex process. Most models focused on the faithful in vitro restoration of T cell differentiation and selection. In contrast, suitable in vitro models emulating the developmental pathways of the two major thymic epithelial cell lineages--cortical thymic epithelial cells and medullary thymic epithelial cells (mTECs)--are yet to be developed. In this regard, lack of an in vitro model mimicking the developmental biology of the mTEC lineage has hampered the molecular analysis of the so-called "promiscuous expression" of tissue-restricted genes, a key property of terminally differentiated mTECs. Based on the close biological relationship between the skin and thymus epithelial cell compartments, we adapted a three-dimensional organotypic coculture model, originally developed to provide a bona fide in vitro dermal equivalent, for the culture of isolated mTECs. This three-dimensional model preserves key features of mTECs: proliferation and terminal differentiation of CD80(lo), Aire(-) mTECs into CD80(hi), Aire(+) mTECs; responsiveness to RANKL; and sustained expression of FoxN1, Aire, and tissue-restricted genes in CD80(hi) mTECs. This in vitro culture model should facilitate the identification of molecular components and pathways involved in mTEC differentiation in general and in promiscuous gene expression in particular.

Transcriptomic diversity in human medullary thymic epithelial cells.

The induction of central T cell tolerance in the thymus depends on the presentation of peripheral self-epitopes by medullary thymic epithelial cells (mTECs). This promiscuous gene expression (pGE) drives mTEC transcriptomic diversity, with non-canonical transcript initiation, alternative splicing, and expression of endogenous retroelements (EREs) representing important but incompletely understood contributors. Here we map the expression of genome-wide transcripts in immature and mature human mTECs using high-throughput 5' cap and RNA sequencing. Both mTEC populations show high splicing entropy, potentially driven by the expression of peripheral splicing factors. During mTEC maturation, rates of global transcript mis-initiation increase and EREs enriched in long terminal repeat retrotransposons are up-regulated, the latter often found in proximity to differentially expressed genes. As a resource, we provide an interactive public interface for exploring mTEC transcriptomic diversity. Our findings therefore help construct a map of transcriptomic diversity in the healthy human thymus and may ultimately facilitate the identification of those epitopes which contribute to autoimmunity and immune recognition of tumor antigens.

Promiscuous gene expression patterns in single medullary thymic epithelial cells argue for a stochastic mechanism.

Promiscuous expression of tissue-restricted autoantigens in medullary thymic epithelial cells (mTECs) imposes central T cell tolerance. The molecular regulation of this unusual gene expression is not understood, in particular its delineation from cell lineage-specific gene expression control remains unclear. Here, we compared the expression profile of the casein gene locus in mTECs and mammary gland epithelial cells by single cell PCR. Mammary gland cells showed highly correlated intra- and interchromosomal coexpression of milk proteins (the casein genes, lactalbumin-alpha and whey acidic protein) and one of its transcriptional regulators (Elf5). In contrast, coexpression of these genes in mature CD80(hi) mTECs was rarely observed and no pattern of gene expression in individual mTECs was discernible. The apparent stochastic expression pattern of genes within the casein locus, the lower mRNA levels compared with mammary gland cells in conjunction with frequent coexpression of insulin in single mTECs clearly delineates the molecular mechanism(s) of promiscuous gene expression from cell lineage-specific gene control.

CD8+ T Cells Variably Recognize Native Versus Citrullinated GRP78 Epitopes in Type 1 Diabetes.

In type 1 diabetes, autoimmune ß-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Discovery of Hydroxyamidine Based Inhibitors of IDO1 for Cancer Immunotherapy with Reduced Potential for Glucuronidation.

Following the impressive success of checkpoint inhibitors in the treatment of cancer, combinations of IDO1 inhibitors with PD-1/PD-L1 antibodies are in clinical development aiming to increase response rates. Using the hydroxyamidine pharmacophore of the IDO1 inhibitor INCB14943 as a starting point for the design of new inhibitors, the potential shortcomings of extensive hydroxyamidine glucuronidation in humans was addressed. Compounds were optimized using a stability assay with recombinant UGT1A9 enzyme together with the measurement of glucuronide formation in human hepatocytes. Optimized analog 24 showed cellular and biochemical IDO1 IC50 values in the low nanomolar range, a suitable in vitro ADME/PK profile, and efficacy in an animal model of cancer. In a humanized liver mouse model the lead compound exhibited significantly reduced glucuronidation compared to epacadostat (2).

Peptides Derived From Insulin Granule Proteins Are Targeted by CD8+ T Cells Across MHC Class I Restrictions in Humans and NOD Mice.

The antigenic peptides processed by ß-cells and presented through surface HLA class I molecules are poorly characterized. Each HLA variant (e.g., the most common being HLA-A2 and HLA-A3) carries some peptide-binding specificity. Hence, features that, despite these specificities, remain shared across variants may reveal factors favoring ß-cell immunogenicity. Building on our previous description of the HLA-A2/A3 peptidome of ß-cells, we analyzed the HLA-A3-restricted peptides targeted by circulating CD8+ T cells. Several peptides were recognized by CD8+ T cells within a narrow frequency (1-50/106), which was similar in donors with and without type 1 diabetes and harbored variable effector/memory fractions. These epitopes could be classified as conventional peptides or neoepitopes, generated either via peptide cis-splicing or mRNA splicing (e.g., secretogranin-5 [SCG5]-009). As reported for HLA-A2-restricted peptides, several epitopes originated from ß-cell granule proteins (e.g., SCG3, SCG5, and urocortin-3). Similarly, H-2Kd-restricted CD8+ T cells recognizing the murine orthologs of SCG5, urocortin-3, and proconvertase-2 infiltrated the islets of NOD mice and transferred diabetes into NOD/scid recipients. The finding of granule proteins targeted in both humans and NOD mice supports their disease relevance and identifies the insulin granule as a rich source of epitopes, possibly reflecting its impaired processing in type 1 diabetes.

An efficient protocol for in vivo labeling of proliferating epithelial cells.

The study of organogenesis, tissue-homeostasis and regeneration requires the precise assessment of in vivo cell proliferation. To this end a host of methods have been developed to detect and quantify DNA synthesis in proliferating cells. These include cell labeling with various nucleotide analogues and fluorescence reporter-based animal models with each method presenting its idiosyncratic shortcomings. Quantitative assessment of epithelial cell turnover has been partly hampered due to their variable and limited in vivo accessibility and the requirement for harsher isolation procedures to procure single cells for FACS analysis. Here, we report a reliable protocol to study in vivo cell proliferation of epithelial cells in mice by repeatedly injecting EdU intravenously for an extended 12-day period. EdU incorporation was quantitated ex vivo by FACS after tissue dissociation in order to obtain single epithelial cell suspensions. As a lead population, we analyzed thymic epithelial cells (TECs), where we were able to label compartmentalized TEC subsets to saturation without apparent toxic effects on the thymus architecture or stress-sensitive TEC lineage differentiation. The data is in concordance with the prevailing model of medullary TEC terminal differentiation that includes the post-Aire stage. The same protocol was successfully applied to epithelial cells of various other organs - skin, lymph node, kidney and small intestine - tissues with widely varying frequencies and rates of proliferating epithelial cells.

Islet-reactive CD8+ T cell frequencies in the pancreas, but not in blood, distinguish type 1 diabetic patients from healthy donors.

The human leukocyte antigen-A2 (HLA-A2)-restricted zinc transporter 8186-194 (ZnT8186-194) and other islet epitopes elicit interferon-γ secretion by CD8+ T cells preferentially in type 1 diabetes (T1D) patients compared with controls. We show that clonal ZnT8186-194-reactive CD8+ T cells express private T cell receptors and display equivalent functional properties in T1D and healthy individuals. Ex vivo analyses further revealed that CD8+ T cells reactive to ZnT8186-194 and other islet epitopes circulate at similar frequencies and exhibit a predominantly naïve phenotype in age-matched T1D and healthy donors. Higher frequencies of ZnT8186-194-reactive CD8+ T cells with a more antigen-experienced phenotype were detected in children versus adults, irrespective of disease status. Moreover, some ZnT8186-194-reactive CD8+ T cell clonotypes were found to cross-recognize a Bacteroides stercoris mimotope. Whereas ZnT8 was poorly expressed in thymic medullary epithelial cells, variable thymic expression levels of islet antigens did not modulate the peripheral frequency of their cognate CD8+ T cells. In contrast, ZnT8186-194-reactive cells were enriched in the pancreata of T1D patients versus nondiabetic and type 2 diabetic individuals. Thus, islet-reactive CD8+ T cells circulate in most individuals but home to the pancreas preferentially in T1D patients. We conclude that the activation of this common islet-reactive T cell repertoire and progression to T1D likely require defective peripheral immunoregulation and/or a proinflammatory islet microenvironment.

Conventional and Neo-antigenic Peptides Presented by ß Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors.

Although CD8+ T-cell-mediated autoimmune ß cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by ß cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known ß cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by ß cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.

The PTI1-like kinase ZmPti1a from maize (Zea mays L.) co-localizes with callose at the plasma membrane of pollen and facilitates a competitive advantage to the male gametophyte.

BACKGROUND: The tomato kinase Pto confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato in a gene for gene manner. Upon recognition of specific avirulence factors the Pto kinase activates multiple signal transduction pathways culminating in induction of pathogen defense. The soluble cytoplasmic serine/threonine kinase Pti1 is one target of Pto phosphorylation and is involved in the hypersensitive response (HR) reaction. However, a clear role of Pti1 in plant pathogen resistance is uncertain. So far, no Pti1 homologues from monocotyledonous species have been studied. RESULTS: Here we report the identification and molecular analysis of four Pti1-like kinases from maize (ZmPti1a, -b, -c, -d). These kinase genes showed tissue-specific expression and their corresponding proteins were targeted to different cellular compartments. Sequence similarity, expression pattern and cellular localization of ZmPti1b suggested that this gene is a putative orthologue of Pti1 from tomato. In contrast, ZmPti1a was specifically expressed in pollen and sequestered to the plasma membrane, evidently owing to N-terminal modification by myristoylation and/or S-acylation. The ZmPti1a:GFP fusion protein was not evenly distributed at the pollen plasma membrane but accumulated as an annulus-like structure which co-localized with callose (1,3-beta-glucan) deposition. In addition, co-localization of ZmPti1a and callose was observed during stages of pollen mitosis I and pollen tube germination. Maize plants in which ZmPti1a expression was silenced by RNA interference (RNAi) produced pollen with decreased competitive ability. Hence, our data provide evidence that ZmPti1a plays an important part in a signalling pathway that accelerates pollen performance and male fitness. CONCLUSION: ZmPti1a from maize is involved in pollen-specific processes during the progamic phase of reproduction, probably in crucial signalling processes associated with regions of callose deposition. Pollen-sporophyte interactions and pathogen induced HR show certain similarities. For example, HR has been shown to be associated with cell wall reinforcement through callose deposition. Hence, it is hypothesized that Pti1 kinases from maize act as general components in evolutionary conserved signalling processes associated with callose, however during different developmental programs and in different tissue types.

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance. Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80(lo), Aire-negative into CD80(hi), Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80(hi) mTECs.