Mesenchymal cell replacement corrects thymic hypoplasia in murine models of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations, thymic hypoplasia, hypoparathyroidism, and/or cardiac defects. Thymic hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymic hypoplasia in 22q11.2DS remain unknown. Comparison of embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed proportions of mesenchymal, epithelial, and hematopoietic cell types similar to those of control thymuses. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymic mesenchymal cells with normal ones restored tissue growth. Comparative single-cell RNA-Seq of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 cell line. The transcripts affected included those for extracellular matrix proteins, consistent with the increased collagen deposition we observed in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymic tissue expansion for hypoplastic lobes. In colony-forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, in contrast to the normal growth of thymic epithelial cells. These findings suggest that mesenchymal cells were causal to the small embryonic thymuses in the 22q11.2DS mouse models, which was correctable by substitution with normal mesenchyme.

FOXN1 compound heterozygous mutations cause selective thymic hypoplasia in humans.

We report on 2 patients with compound heterozygous mutations in forkhead box N1 (FOXN1), a transcription factor essential for thymic epithelial cell (TEC) differentiation. TECs are critical for T cell development. Both patients had a presentation consistent with T-/loB+NK+ SCID, with normal hair and nails, distinct from the classic nude/SCID phenotype in individuals with autosomal-recessive FOXN1 mutations. To understand the basis of this phenotype and the effects of the mutations on FOXN1, we generated mice using CRISPR-Cas9 technology to genocopy mutations in 1 of the patients. The mice with the Foxn1 compound heterozygous mutations had thymic hypoplasia, causing a T-B+NK+ SCID phenotype, whereas the hair and nails of these mice were normal. Characterization of the functional changes due to the Foxn1 mutations revealed a 5-amino acid segment at the end of the DNA-binding domain essential for the development of TECs but not keratinocytes. The transcriptional activity of this Foxn1 mutant was partly retained, indicating a region that specifies TEC functions. Analysis of an additional 9 FOXN1 mutations identified in multiple unrelated patients revealed distinct functional consequences contingent on the impact of the mutation on the DNA-binding and transactivation domains of FOXN1.

MIR205HG Is a Long Noncoding RNA that Regulates Growth Hormone and Prolactin Production in the Anterior Pituitary.

MicroRNAs (miRNAs) are processed from primary miRNA transcripts (pri-miRNAs), many of which are annotated as long noncoding RNAs (lncRNAs). We assessed whether MIR205HG, the host gene for miR-205, has independent functions as an lncRNA. Comparing mice with targeted deletions of MIR205HG and miR-205 revealed a functional role for the lncRNA in the anterior pituitary. Mice lacking MIR205HG had a temporal reduction in Pit1, growth hormone, and prolactin. This was mediated, in part, through the ability of this lncRNA to bind and regulate the transcriptional activity of Pit1 in conjunction with Zbtb20. Knockdown of MIR205HG in lactotropes decreased the expression of Pit1, Zbtb20, prolactin, and growth hormone, while its overexpression enhanced the levels of these transcripts. The effects of MIR205HG on the pituitary were independent of miR-205. The data support a role for MIR205HG as an lncRNA that regulates growth hormone and prolactin production in the anterior pituitary.

The Genetics and Epigenetics of 22q11.2 Deletion Syndrome.

Chromosome 22q11.2 deletion syndrome (22q11.2del) is a complex, multi-organ disorder noted for its varying severity and penetrance among those affected. The clinical problems comprise congenital malformations; cardiac problems including outflow tract defects, hypoplasia of the thymus, hypoparathyroidism, and/or dysmorphic facial features. Additional clinical issues that can appear over time are autoimmunity, renal insufficiency, developmental delay, malignancy and neurological manifestations such as schizophrenia. The majority of individuals with 22q11.2del have a 3 Mb deletion of DNA on chromosome 22, leading to a haploinsufficiency of ~106 genes, which comprise coding RNAs, noncoding RNAs, and pseudogenes. The consequent haploinsufficiency of many of the coding genes are well described, including the key roles of T-box Transcription Factor 1 (TBX1) and DiGeorge Critical Region 8 (DGCR8) in the clinical phenotypes. However, the haploinsufficiency of these genes alone cannot account for the tremendous variation in the severity and penetrance of the clinical complications among those affected. Recent RNA and DNA sequencing approaches are uncovering novel genetic and epigenetic differences among 22q11.2del patients that can influence disease severity. In this review, the role of coding and non-coding genes, including microRNAs (miRNA) and long noncoding RNAs (lncRNAs), will be discussed in relation to their bearing on 22q11.2del with an emphasis on TBX1.

MicroRNA-205 Maintains T Cell Development following Stress by Regulating Forkhead Box N1 and Selected Chemokines.

The thymus, an organ responsible for T cell development, is one of the more stress-sensitive tissues in the body. Stress, in the form of infections, radiation exposure, and steroids, impairs thymic epithelial cell (TEC) functions and induces the programmed cell death of immature thymocytes. MicroRNAs are small noncoding RNAs involved in tissue repair and homeostasis, with several supporting T cell development. We report that miR-205, an epithelial-specific miR, maintains thymopoiesis following inflammatory perturbations. Thus, the activation of diverse pattern recognition receptors in mice causes a more severe thymic hypoplasia and delayed T cell recovery when miR-205 is conditionally ablated in TECs. Gene expression comparisons in the TECs with/without miR-205 revealed a significant differential regulation of chemokine/chemokine receptor pathways, antigen processing components, and changes in the Wnt signaling system. This was partly a consequence of reduced expression of the transcriptional regulator of epithelial cell function, Forkhead Box N1 (Foxn1), and its two regulated targets, stem cell factor and ccl25, following stress. miR-205 mimics supplemented into miR-205-deficient fetal thymic organ cultures restored Foxn1 expression along with ccl25 and stem cell factor A number of putative targets of miR-205 were up-regulated in TECs lacking miR-205, consistent with an important role for this miR in supporting T cell development in response to stress.

Agonist antibody activates death receptor 6 downstream signaling involving TRADD recruitment.

Death receptor 6 (DR6) is a member of the death domain-containing receptors that belong to the TNFR superfamily. To date, the ligand for DR6 is still not clearly defined. Here, we developed a functional agonist monoclonal antibody (DQM3) against DR6, which bound to the first cysteine-rich domain. Importantly, DR6 signaling could be clearly activated by DQM3, which was dependent on its intracellular death domain. In addition, we demonstrated that the association between DR6 and TRADD was enhanced upon DQM3 stimulation and TRADD was involved in DR6-induced signaling activation. Taken together, our findings provide new insight into a novel mechanism by which DR6 induces downstream signaling in response to an agonist antibody.

CD2AP/SHIP1 complex positively regulates plasmacytoid dendritic cell receptor signaling by inhibiting the E3 ubiquitin ligase Cbl.

The human plasmacytoid dendritic cell (pDC) receptor BDCA2 forms a complex with the adaptor FcεR1γ to activate an ITAM-signaling cascade. BDCA2 receptor signaling negatively regulates the TLR7/9-mediated type 1 IFN responses in pDCs, which may play a key role in controlling self-DNA/RNA-induced autoimmunity. We report in this article that CD2-associated adaptor protein (CD2AP), which is highly expressed in human pDCs, positively regulates BDCA2/FcεR1γ receptor signaling. By immunoprecipitation and mass spectrometry analyses, we found that CD2AP bound to SHIP1. Knockdown of CD2AP or SHIP1 reduced the BDCA2/FcεR1γ-mediated ITAM signaling and blocked its inhibition of TLR9-mediated type 1 IFN production. Knockdown of CD2AP or SHIP1 also enhanced the ubiquitination and degradation of Syk and FcεR1γ that was mediated by the E3 ubiquitin ligase Cbl. This led us to discover that, upon BDCA2 cross-linking, the CD2AP/SHIP1 complex associated with Cbl and inhibited its E3 ubiquitin ligase activity. In human primary pDCs, cross-linking of the BDCA2/FcεR1γ complex induced the recruitment of the CD2AP/SHIP1/Cbl complex to the plasma membrane of pDCs, where it colocalized with the BDCA2/FcεR1γ complex. Therefore, CD2AP positively regulates BDCA2/FcεR1γ signaling by forming a complex with SHIP1 to inhibit the E3 ubiquitin ligase Cbl.

Preferential depletion of CD2(low) plasmacytoid dendritic cells in HIV-infected subjects.

Plasmacytoid dendritic cells (pDCs) are decreased in number and are functionally impaired in HIV act reasons for pDCs depletion are still unknown. It was recently reported that pDCs can be divided into two functionally distinct populations based on their CD2 expression level. To determine how the CD2(high) and CD2(low) populations are affected by HIV infection, we analyzed their frequencies in the peripheral blood of HIV-infected subjects and healthy controls. We found that the CD2(low) pDC subset was preferentially depleted in infected individuals. The frequency of CD2(low) pDCs correlated with the CD4(+) T-cell count but not with the plasma viral load. This finding furthers our understanding of the causes and consequences of pDC depletion during HIV infection.

Plasmacytoid dendritic cells in antiviral immunity and autoimmunity.

Plasmacytoid dendritic cells (pDCs) represent a unique and crucial immune cell population capable of producing large amounts of type I interferons (IFNs) in response to viral infection. The function of pDCs as the professional type I IFN-producing cells is linked to their selective expression of Toll-like receptor 7 (TLR7) and TLR9, which sense viral nucleic acids within the endosomal compartments. Type I IFNs produced by pDCs not only directly inhibit viral replication but also play an essential role in linking the innate and adaptive immune system. The aberrant activation of pDCs by self nucleic acids through TLR signaling and the ongoing production of type I IFNs do occur in some autoimmune diseases. Therefore, pDC may serve as an attractive target for therapeutic manipulations of the immune system to treat viral infectious diseases and autoimmune diseases.

Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction.

Plasmacytoid dendritic cells (pDCs) produce copious type I interferon (IFN) upon sensing nucleic acids through Toll-like receptor (TLR) 7 and TLR9. Uncontrolled pDC activation and IFN production are implicated in lymphopenia and autoimmune diseases; therefore, a mechanism controlling pDC IFN production is essential. Human pDCs specifically express an orphan receptor, immunoglobulin-like transcript 7 (ILT7). Here, we discovered an ILT7 ligand expressed by human cell lines and identified it as bone marrow stromal cell antigen 2 (BST2; CD317). BST2 directly binds to purified ILT7 protein, initiates signaling via the ILT7-FcepsilonRIgamma complex, and strongly inhibits production of IFN and proinflammatory cytokines by pDCs. Readily induced by IFN and other proinflammatory cytokines, BST2 may modulate the human pDC's IFN responses through ILT7 in a negative feedback fashion.