Quantification of dendritic cell subsets in human thymus tissues of various ages

Background Dendritic cells (DCs) in the thymus are involved in central tolerance formation, but they also have other functions in the thymus, such as pathogen recognition. The density changes of human thymic DCs have been hardly investigated. In this study, human thymus samples of various ages were collected for tissue sectioning and staining. The thymic cortex and medulla area as well as the densities of various subsets of thymic DCs were calculated. Results All common DC subsets were found in the human thymus of various ages. Most DCs had accumulated in the human thymic epithelial space, especially the medulla. We also found that the human thymic cortex had atrophied relatively faster than the medulla, which led to a gradual increase of the area ratio of the medulla to cortex with the increase of age. The densities of DC subsets in the human thymus showed various changes with increasing age, which contributed to the composition changes of DC subsets. The density of plasmacytoid DCs (pDCs) in the human thymus had increased gradually with aging, which suggested that pDCs plays another essential role in the thymus in addition to central tolerance. Conclusions Inconsistent with the shrinking of the epithelial space in the thymus, the densities of DC subsets in the epithelial space of the thymus are maintained at a constant level with aging to preserve highly efficient autoreactive thymocyte screening. An increasing density of the thymic pDCs with aging implies an extra function of DCs in the thymus beyond central tolerance.

Pharmacological Targeting of PI3K-Dependent Central Tolerance Mechanisms in Refractory Pre-Germinal Center B-Cell Malignancies

Rationale: About 75% of newly formed B-cells are autoreactive and express potentially harmful autoantibodies (Wardemann 2003). Hence, a powerful mechanism, termed central tolerance, is in place to eliminate millions of newly formed autoreactive B-cells every day. Results: B-ALL, mantle cell lymphoma (MCL) and unmutated chronic lymphocytic leukemia (U-CLL) originate from early, pre-germinal center (pre-GC) stages of B-cell development that are subject to negative B-cell selection and central tolerance mechanisms. While designed to eliminate autoreactive clones during early B-cell development, we recently discovered that B-ALL, MCL and U-CLL fully retained sensitivity to central tolerance mechanisms, which are triggered by persistent PI3K-hyperactivation. PI3K-signaling code to distinguish between normal and pathological signaling. Studying short transient pulses and chronic activation of PI3K-signaling, we discovered that pre-GC B-cells have evolved a "PI3K-signaling code" to distinguish between normal B-cell activation by antigen and pathological signaling: thereby, antigen encounter induces a short transient pulse of PI3K-activation which promotes survival and proliferation. Conversely, persistent activation of PI3K-activation reflects pathological signaling, either from an autoreactive B-cell receptor (BCR) or a transforming oncogene. Pre-GC B-cell malignancies are exempt from oncogenic PI3K-lesions. PI3K-lesions in cancer result in permanent hyperactivation as in autoreactive B-cells. The PI3K pathway is targeted by oncogenic lesions in ~25% of human cancer. The phosphatases PTEN, SHIP1 and PP2A function as negative regulators of PI3K signaling and are frequently mutated in a broad range of cancers and also occur in some GC- and post-GC lymphomas (e.g. Burkitt's, DLBCL). However, our analysis in six clinical cohorts revealed that pre-GC B-cell malignancies, including B-ALL, MCL and U-CLL critically depend on PTEN, SHIP1 and PP2A function and do not tolerate persistent hyperactivation of PI3K-signaling for more than three hours. Loss-of-function mutations of these phosphatases and activating PI3K lesions were not detected in large clinical cohorts of patients with B-ALL, MCL and CLL. Likewise, phosphorylation of AKT-S473, reflecting PI3K signaling strength, is elevated throughout multiple cancer types including post-GC DLBCL, but not in B-ALL and MCL. This is in line with previous work demonstrating that inherited mutations that cause PI3K-activation predispose to various cancers but cause profound defects in human B-lymphopoiesis (Fruman 2014). Pharmacological targeting of PI3K-dependent central tolerance mechanisms. We tested the hypothesis that PI3K-hyperactivation represents a unique vulnerability in pre-GC B-cell tumors including B-ALL, MCL and U-CLL. Sensitivity to PI3K-hyperactivation of reflects their pre-GC origin and central tolerance mechanisms during early B-cell development that are designed to eliminate autoreactive B-cells based on hyperactive PI3K-signaling. For this reason, we tested pharmacological PI3K-hyperactivation as a novel strategy to selectively target pre-GC B-cell malignancies. To this end, we tested 144 compounds for their ability to engage PI3K-dependent central tolerance mechanism in B-ALL, MCL and CLL. Small molecule inhibitors of SHIP1 (3AC, K118), PTEN (SF-1670), PP2A (LB-100) and a direct PI3K-agonist (SC79) achieved strong phosphorylation of known PI3K-substrates (AKT, S6K) in vitro and prolonged overall survival in NSG mice transplanted with refractory B-ALL and MCL PDX in vivo. Conclusions and future directions: Current treatment regimens (kinase-inhibitor paradigm) use agents that apply selective pressure in one direction (e.g., PI3K-inhibitors; BCR-ABL1, SYK- or BTK-inhibitors). Here, we are pursuing a new concept (central tolerance paradigm) based on sequential treatment regimens that alternate between kinase-inhibitors (e.g., dasatinib, ibrutinib, idelalisib) and PI3K-hyperactivation (3AC, K118, LB100). By sequentially applying selective pressures in opposite directions, our approach will subvert clonal evolution and selection for drug-resistant mutants. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The Multifaceted Roles of B Cells in the Thymus: From Immune Tolerance to Autoimmunity

The thymus is home to a significant number of resident B cells which possess several unique characteristics regarding their origin, phenotype and function. Evidence shows that they originate both from precursors that mature intrathymically and as the entry of recirculating mature B cells. Under steady-state conditions they exhibit hallmark signatures of activated B cells, undergo immunoglobulin class-switch, and express the Aire transcription factor. These features are imprinted within the thymus and enable B cells to act as specialized antigen-presenting cells in the thymic medulla that contribute negative selection of self-reactive T cells. Though, most studies have focused on B cells located in the medulla, a second contingent of B cells is also present in non-epithelial perivascular spaces of the thymus. This latter group of B cells, which includes memory B cells and plasma cells, is not readily detected in the thymus of infants or young mice but gradually accumulates during normal aging. Remarkably, in many autoimmune diseases the thymus suffers severe structural atrophy and infiltration of B cells in the perivascular spaces, which organize into follicles similar to those typically found in secondary lymphoid organs. This review provides an overview of the pathways involved in thymic B cell origin and presents an integrated view of both thymic medullary and perivascular B cells and their respective physiological and pathological roles in central tolerance and autoimmune diseases.

Specialized transendothelial dendritic cells mediate thymic T-cell selection against blood-borne macromolecules

T cells undergo rigorous selection in the thymus to ensure self-tolerance and prevent autoimmunity, with this process requiring innocuous self-antigens (Ags) to be presented to thymocytes. Self-Ags are either expressed by thymic stroma cells or transported to the thymus from the periphery by migratory dendritic cells (DCs); meanwhile, small blood-borne peptides can access the thymic parenchyma by diffusing across the vascular lining. Here we describe an additional pathway of thymic Ag acquisition that enables circulating antigenic macromolecules to access both murine and human thymi. This pathway depends on a subset of thymus-resident DCs, distinct from both parenchymal and circulating migratory DCs, that are positioned in immediate proximity to thymic microvessels where they extend cellular processes across the endothelial barrier into the blood stream. Transendothelial positioning of DCs depends on DC-expressed CX3CR1 and its endothelial ligand, CX3CL1, and disrupting this chemokine pathway prevents thymic acquisition of circulating proteins and compromises negative selection of Ag-reactive thymocytes. Thus, transendothelial DCs represent a mechanism by which the thymus can actively acquire blood-borne Ags to induce and maintain central tolerance.

Thymic B Cells as a New Player in the Type 1 Diabetes Response

Type 1 diabetes (T1d) results from a sustained autoreactive T and B cell response towards insulin-producing β cells in the islets of Langerhans. The autoreactive nature of the condition has led to many investigations addressing the genetic or cellular changes in primary lymphoid tissues that impairs central tolerance- a key process in the deletion of autoreactive T and B cells during their development. For T cells, these studies have largely focused on medullary thymic epithelial cells (mTECs) critical for the effective negative selection of autoreactive T cells in the thymus. Recently, a new cellular player that impacts positively or negatively on the deletion of autoreactive T cells during their development has come to light, thymic B cells. Normally a small population within the thymus of mouse and man, thymic B cells expand in T1d as well as other autoimmune conditions, reside in thymic ectopic germinal centres and secrete autoantibodies that bind selective mTECs precipitating mTEC death. In this review we will discuss the ontogeny, characteristics and functionality of thymic B cells in healthy and autoimmune settings. Furthermore, we explore how in silico approaches may help decipher the complex cellular interplay of thymic B cells with other cells within the thymic microenvironment leading to new avenues for therapeutic intervention.

The Immune System in Duchenne Muscular Dystrophy Pathogenesis

Growing evidence demonstrates the crosstalk between the immune system and the skeletal muscle in inflammatory muscle diseases and dystrophic conditions such as Duchenne Muscular Dystrophy (DMD), as well as during normal muscle regeneration. The rising of inflammation and the consequent activation of the immune system are hallmarks of DMD: several efforts identified the immune cells that invade skeletal muscle as CD4+ and CD8+ T cells, Tregs, macrophages, eosinophils and natural killer T cells. The severity of muscle injury and inflammation dictates the impairment of muscle regeneration and the successive replacement of myofibers with connective and adipose tissue. Since immune system activation was traditionally considered as a consequence of muscular wasting, we recently demonstrated a defect in central tolerance caused by thymus alteration and the presence of autoreactive T-lymphocytes in DMD. Although the study of innate and adaptive immune responses and their complex relationship in DMD attracted the interest of many researchers in the last years, the results are so far barely exhaustive and sometimes contradictory. In this review, we describe the most recent improvements in the knowledge of immune system involvement in DMD pathogenesis, leading to new opportunities from a clinical point-of-view.

P03.07 Analysis of scRNAseq from the human thymus nominates genes potentially missing from central tolerance of cytotoxic T cells

BackgroundDuring thymic development, cytotoxic T cells that can bind to and attack self antigens undergo negative selection thus preventing damage to the self tissues. The sparse medullar thymic epithelial cells (mTECs) present in the thymus are responsible for presenting self antigens to T cells so that they can trigger apoptosis or differentiation into non-cytotoxic lineages if they bind too strongly.Materials and MethodsUnderstanding gene expression in mTECs is essential for understanding the shape of the human T cell receptor repertoire, which is key for current and emerging cancer immunotherapies. Recent availability of human thymus single cell RNAseq (scRNAseq) data provides an extremely high-resolution view into the pattern of expression within this critical cell type. To determine which epitopes have had to opportunity to be presented during T cell negative selection, we analyzed the human thymus scRNAseq dataset to establish which genes are expressed in mTECs and therefore subject to central tolerance.ResultsThe coverage of the whole transcriptome of a particular cell is generally sparse. It is therefore difficult to understand basic features of individual cells or cell types such as how many genes are expressed. We used cell- and read-level subsampling to estimate whether a sufficient number of cells and reads had been captured to support categorizing a gene as non-expressed in mTECs. We also examined the expression of the genes not expressed in mTECs in other healthy tissues, and found their expression was almost exclusively restricted to the testis (an immune-privileged site) and the liver (a site of peripheral tolerance)ConclusionsAltogether, these analyses establish a strategy for determining if a data set has sufficient depth to estimate the total number of genes expressed and secondly define a key list of genes that are not expressed during central tolerization of T cells, which represent a compelling list of possible cancer immunotherapy targets.Disclosure InformationL. Blumenberg: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals. G. Atwal: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals. A. Dhanik: A. Employment (full or part-time); Significant; Regeneron Pharmaceuticals.

A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer

Medullary thymic epithelial cells (mTECs) which produce and present self-antigens are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with specific subset of DCs. Using several relevant Cre-based mouse models controlling the expression of fluorescent proteins, we found that in regards to CAT, each subset of thymic DCs preferentially targets distinct mTEC subset(s) and importantly, XCR1+ activated DCs represented the most potent subset in CAT. Interestingly, one thymic DC can acquire antigen repetitively and of these, monocyte-derived DCs (moDC) were determined to be the most efficient in repetitive CAT. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

Mechanistic dissection of dominant AIRE mutations in mouse models reveals AIRE autoregulation

The autoimmune regulator (AIRE) is essential for the establishment of central tolerance and prevention of autoimmunity. Interestingly, different AIRE mutations cause autoimmunity in either recessive or dominant-negative manners. Using engineered mouse models, we establish that some monoallelic mutants, including C311Y and C446G, cause breakdown of central tolerance. By using RNAseq, ATACseq, ChIPseq, and protein analyses, we dissect the underlying mechanisms for their dominancy. Specifically, we show that recessive mutations result in a lack of AIRE protein expression, while the dominant mutations in both PHD domains augment the expression of dysfunctional AIRE with altered capacity to bind chromatin and induce gene expression. Finally, we demonstrate that enhanced AIRE expression is partially due to increased chromatin accessibility of the AIRE proximal enhancer, which serves as a docking site for AIRE binding. Therefore, our data not only elucidate why some AIRE mutations are recessive while others dominant, but also identify an autoregulatory mechanism by which AIRE negatively modulates its own expression.