Thymocyte Differentiation Research Articles

Mechanisms of hormonal control of thymic differentiation process in the aspect of pregnancy

The effect of hormones (chorionic gonadotropin, estriol, leptin, ghrelin, kisspeptin) in the regulation of cell differentiation in the thymus was studied, in concentrations being comparable with the content in peripheral blood during pregnancy. It was established that the studied hormones regulate the functional activity of myeloid and plasmacytoid thymic dendritic cells in different directions, thus mediating the regulatory effects on the differentiation of thymocytes in contact with them. Hormones modulate the expression of the co-stimulatory activation and inhibitory molecules on myeloid and plasmacytoid thymic dendritic cells, as well as the activity of the enzyme indolamine-2,3-dioxigenase in general, increasing the tolerogenic potential of thymus dendritic cells. Hormone-influenced myeloid and plasmocytoid dendritic cells of the thymus have opposite effects on the formation of natural regulatory subpopulations of thymocytes (iNKT, nTreg, Th17), contributing to the successful development of physiological pregnancy. The effects of the studied hormones on the differentiation of αβ and δT cells in the thymus are mediated by the influence of hormones on both types of thymus dendritic cells, which complements the understanding of the regulatory mechanisms of steroid-induced thymus involution during pregnancy. Thus, the hormones produced by the placenta during pregnancy regulate the maturation and functional activity of thymus dendritic cells, modulating their ability to influence the differentiation of regulatory and effector populations of thymocytes. The obtained results reveal new mechanisms of hormonal regulation of the thymic differentiation of cells of the immune system which can be realized at different periods of pregnancy.

Lectin panel screening for evaluating of murine thymocytes apoptosis stages

The aim of the study was to investigate the interaction of lectins with various populations of maturing murine T-lymphocytes, as well as with thymocytes at different stages of apoptosis.
 Materials and methods. Thymocyte typing of 80 CBA mice was performed by flow cytometry. The binding of lectins to cells in early and late apoptosis induced by the administration of hydrocortisone was also evaluated.
 Results. The suitability of peanut and Helix pomatia lectins for differentiation of mature and immature mouse thymocytes has been established. 11 lectins bound to living cells, during the transition of cells to the state of early apoptosis, thymocytes were stained with 16 lectins, and upon transition to late apoptosis, 20 of 23 lectins bound to the cells.
 Conclusion. The use of labeled lectins to assess the stage of murine thymocyte apoptosis does not have obvious advantages over existing methods. The degree of binding of all lectins to thymocytes in apoptosis increases as the charge on the membrane decreases and its permeability increases. For typing thymocytes in the early stages of maturation, peanut and Helix pomatia lectins can be used. Snowdrop and amaryllis lectins are not suitable for differentiation of thymocytes by maturity.

CD Maps - Dynamic Profiling of CD1 to CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets and their malignant counterparts. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-100 (n=110) on 47 immune cell subsets from blood, thymus and tonsil using an 8-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABC) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as, to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases. CD Maps project is supported with reagents from BD Biosciences, BioLegend and Exbio. TK is supported by Ministry of Health Czech Republic grant 15-26588A and LO1604. KF is supported by Ministry of Health Czech Republic grant NV18-08-00385. Disclosures No relevant conflicts of interest to declare.

Idh2R140Q Cooperates with NHD13 to Induce Immature T Cell Leukemia By Targeting Early T Cell Progenitors or DN2 Thymocytes

Introduction Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequently observed in patients with acute myeloid leukemia, with IDH2 mutations reported in 9- 19% of AML cases. IDH2 mutation leads to loss of normal enzymatic function and accumulation of 2-hydroxyglutarate (2-HG) through a newly gained enzyme activity. The most common IDH mutation in AML patients involves IDH2 R140. Transgenic mice that expressed an Idh2R140Q mutant in all hematopoietic tissues did not develop leukemia, nor was there a significant difference between overall survival of Idh2R140Q mice compared to wildtype mice. These findings were consistent with those from other investigators, which also suggested that expression of mutant IDH2 was not sufficient for development of leukemia, and that collaborating mutations are necessary. A recent report of whole exome sequence (WES) from mouse models of hematopoietic malignancies identified recurrent Idh1R132H mutations in NUP98-HOXD13 (NHD13) driven AML. Given that Idh1 R132 is paralogous to IDH2 R140, we considered the possibility that an Idh2R140Q mutation would collaborate with an NHD13 transgene and promote leukemic transformation. Therefore, we generated Idh2R140Q/NHD13 transgenic mice by crossing Idh2R140Q with NHD13 mice. Idh2R140Q/NHD13 transgenic mice developed a leukemia at a median of 10 months of age that was similar to human ETP in terms of immunophenotype and additional acquired cooperative mutations in genes such as Pten, N/Kras, Ptpn11, and Sh2b3. Results Gene set enrichment analysis (GSEA) based on RNA-Seq data from Idh2R140Q/NHD13 ETP leukemias and non-ETP T cell leukemias showed that Idh2R140Q/NHD13 ETP gene expression profile correlated well with human ETP leukemic expression profile. An in vitro thymocyte differentiation assay using co-culture of immature double negative (DN) 1 and DN2 thymocytes from Idh2R140Q/NHD13 mice on an OP9-DL1 stromal layer demonstrated a complete block in differentiation to double positive (DP) CD4+CD8+ thymocytes. The Idh2R140Q/NHD13 DN1/2 co-cultured cells arrested at the DN2 stage of differentiation, similar to the in-vivo phenotype of Idh2R140Q/NHD13 leukemia. In addition, Idh2R140Q/NHD13 DN1/2 cells had greater proliferative potential compared to wildtype control. We further observed that Idh2R140Q/NHD13 DN cells would proliferate indefinitely on OP9-DL1 stromal cells, and that treatment of Idh2R140Q/NHD13 thymocytes with AG-221, a potent and selective mutant IDH2 inhibitor, led to a marked decrease in cell proliferation. We developed an in vivo bone marrow transplantation (BMT) model for Idh2R140Q/NHD13 ETP leukemia to assess response to AG-221 in vivo. Primary Idh2R140Q/NHD13 ETP leukemia cells were transplanted into sub-lethally (600 cGy) irradiated recipient mice. This resulted in recipient mice that were anemic and thrombocytopenic with elevated white blood cell counts, suggesting engraftment of acute leukemia. The transplanted, secondary leukemias were consistent with the primary disease immunophenotype by flow cytometry, and tissue histology showed infiltration of blasts into the bone marrow, spleen and perivascular regions of the liver, consistent with disseminated leukemia. Blast cells were positive for both CD3 and myeloperoxidase, further highlighting an ETP phenotype. An assay for clonal T cell receptor beta rearrangement confirmed clonality of recipient leukemia cells identical to the primary leukemia cells. In vivo treatment of Idh2R140Q/NHD13 ETP recipient mice with AG221 showed a significant decrease in leukemic cell expansion compared to control mice treated by gavage with vehicle only; survival data is pending. Conclusion In summary, the IDH2 inhibitor data suggests that targeting the mutant IDH2 in Idh2R140Q/NHD13 leukemic cells can result in a significant decrease in leukemic burden in vivo. Furthermore, the Idh2R140Q/NHD13 primary ETP leukemia BMT mice serves as an excellent model for the study of ETP leukemia development and therapy. In this context, it is important to note that 14% of human ETP show mutations in IDH1/2, and although NUP98 translocations are rare but recurrent events in human ETP ALL, these NUP98 translocations lead to enforced expression of HOXA genes, which is a common event in ETP ALL. Disclosures Aplan: NIH: Patents & Royalties: royalties for the invention of NUP98-HOXD13 .

The Bromodomain Protein 4 Contributes to the Regulation of Alternative Splicing

The bromodomain protein 4 (BRD4) is an atypical kinase and histone acetyl transferase (HAT) that binds to acetylated histones and contributes to chromatin remodeling and early transcriptional elongation. During transcription, BRD4 travels with the elongation complex. Since most alternative splicing events take place co-transcriptionally, we asked if BRD4 plays a role in regulating alternative splicing. We report that distinct patterns of alternative splicing are associated with a conditional deletion of BRD4 during thymocyte differentiation invivo. Similarly, the depletion of BRD4 in Tcell acute lymphoblastic leukemia (T-ALL) cells alters patterns of splicing. Most alternatively spliced events affected by BRD4 are exon skipping. Importantly, BRD4 interacts with components of the splicing machinery, as assessed by both immunoprecipitation (IP) and proximity ligation assays (PLAs), and co-localizes on chromatin with the splicing regulator, FUS. We propose that BRD4 contributes to patterns of alternative splicing through its interaction with the splicing machinery during transcription elongation.

Thy-1 depletion and integrin β3 upregulation-mediated PI3K-Akt-mTOR pathway activation inhibits lung fibroblast autophagy in lipopolysaccharide-induced pulmonary fibrosis

Lipopolysaccharide (LPS)-induced autophagy inhibition in lung fibroblasts is closely associated with the activation of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K-Akt-mTOR) pathway. However, the underlying mechanism remains unknown. In this study, we demonstrated that LPS activated the PI3K-Akt-mTOR pathway and inhibited lung fibroblast autophagy by depleting thymocyte differentiation antigen-1 (Thy-1) and upregulating integrin β3 (Itgb3). Challenge of the human lung fibroblast MRC-5 cell line with LPS resulted in significant upregulation of integrin β3, activation of the PI3K-Akt-mTOR pathway and inhibition of autophagy, which could be abolished by integrin β3 silencing by specific shRNA or treatment with the integrin β3 inhibitor cilengitide. Meanwhile, LPS could inhibit Thy-1 expression accompanied with PI3K-Akt-mTOR pathway activation and lung fibroblast autophagy inhibition; these effects could be prevented by Thy-1 overexpression. Meanwhile, Thy-1 downregulation with Thy-1 shRNA could mimic the effects of LPS, inducing the activation of PI3K-Akt-mTOR pathway and inhibiting lung fibroblast autophagy. Furthermore, protein immunoprecipitation analysis demonstrated that LPS reduced the binding of Thy-1 to integrin β3. Thy-1 downregulation, integrin β3 upregulation and autophagy inhibition were also detected in a mouse model of LPS-induced pulmonary fibrosis, which could be prohibited by intratracheal injection of Thy-1 overexpressing adeno-associated virus (AAV) or intraperitoneal injection of the integrin β3 inhibitor cilengitide. In conclusion, this study demonstrated that Thy-1 depletion and integrin β3 upregulation are involved in LPS-induced pulmonary fibrosis, and may serve as potential therapeutic targets for pulmonary fibrosis.

CD Maps-Dynamic Profiling of CD1-CD100 Surface Expression on Human Leukocyte and Lymphocyte Subsets.

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1–CD100 (n = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

Influence of congenital heart disease-caused hypoxia on intrathymic development of t-lymphocytes in children of first year of life

Aim. To study the influence of hypoxia, caused by congenital heart disease, on the intrathymic development of T-lymphocytes and provision of the organism with T-cellular resource in children with cardiac pathology of different degree of severity.
 Materials and methods. The paper presents the results of complex immunomorphological study of the thymus in children of the first year of life (n = 129) with congenital heart diseases. Hystochemical and immunological methods were used to assess the influence of congenital heart disease-caused hypoxia on the functional status of thymus regarding provision of a child with T-lymphocytes.
 Results. It was established that the degree of severity of congenital heart disease correlated with the degree of suppression of T-lymphocyte pool formation. During the first year of life, proliferative activity of thymocytes (assessed by Ki-67 expression) in the thymus falls, being the reason of decrease in the number of CD3+ thymocytes. In conditions of marked hypoxia (blue type of defect) the process of T-lymphocyte differentiation is inhibited that causes preservation of less mature forms of thymocytes. As a result, hypoxia is the reason of proliferation suppression and thymocyte differentiation, influencing subpopulation composition of the cells. Thus, insufficient quantity of thymic migrants enters the peripheral blood that confirms low functional activity of thymus regarding provision of the organism with T-cellular resource.
 Conclusions. No doubt, it can be the cause of immunodeficient state and reduced formation of adaptive immunity in this category of children.

Intrathymic adeno-associated virus gene transfer rapidly restores thymic function and long-term persistence of gene-corrected T cells

Patients with T-cell immunodeficiencies are generally treated with allogeneic hematopoietic stem cell transplantation, but alternatives are needed for patients without matched donors. An innovative intrathymic gene therapy approach that directly targets the thymus might improve outcomes. We sought to determine the efficacy of intrathymic adeno-associated virus (AAV) serotypes to transduce thymocyte subsets and correct the T-cell immunodeficiency in a zeta-associated protein of 70kDa (ZAP-70)-deficient murine model. AAV serotypes were injected intrathymically into wild-type mice, and gene transfer efficiency was monitored. ZAP-70-/- mice were intrathymically injected with an AAV8 vector harboring the ZAP70 gene. Thymus structure, immunophenotyping, T-cell receptor clonotypes, T-cell function, immune responses to transgenes and autoantibodies, vector copy number, and integration were evaluated. AAV8, AAV9, and AAV10 serotypes all transduced thymocyte subsets after in situ gene transfer, with transduction of up to 5% of cells. Intrathymic injection of an AAV8-ZAP-70 vector into ZAP-70-/- mice resulted in a rapid thymocyte differentiation associated with the development of a thymic medulla. Strikingly, medullary thymic epithelial cells expressing the autoimmune regulator were detected within 10days of gene transfer, correlating with the presence of functional effector and regulatory T-cell subsets with diverse T-cell receptor clonotypes in the periphery. Although thymocyte reconstitution was transient, gene-corrected peripheral T cells harboring approximately 1 AAV genome per cell persisted for more than 40weeks, and AAV vector integration was detected. Intrathymic AAV-transduced progenitors promote a rapid restoration of the thymic architecture, with a single wave of thymopoiesis generating long-term peripheral T-cell function.

Noncoding RNA transcription at enhancers and genome folding in cancer.

Changes of nuclear localization of lineage‐specific genes from a transcriptionally inert to permissive environment are a crucial step in establishing the identity of a cell. Noncoding RNA transcription‐mediated genome folding and activation of target gene expression have been found in a variety of cell types. Noncoding RNA ThymoD (thymocyte differentiation factor) transcription at superenhancers is essential for mouse T‐cell lineage commitment. The cessation of ThymoD transcription abolishes transcription‐mediated demethylation, recruiting looping factors such as the cohesin complex, CCCTC‐binding factor (CTCF), ultimately leading to the phenotype of severe combined immunodeficiency and T‐cell leukemia/lymphoma. In this review, we describe the functional role of RNA polymerase II‐mediated transcription at enhancers and in genome folding. We also highlight the involvement of faulty activation or suppression of enhancer transcription and enhancer‐promoter interaction in cancer development.

A temporal thymic selection switch and ligand binding kinetics constrain neonatal Foxp3+ Treg cell development.

The neonatal thymus generates Foxp3+ regulatory T (tTreg) cells that are critical in controlling immune homeostasis and preventing multiorgan autoimmunity. The role of antigen specificity on neonatal tTreg cell selection is unresolved. Here we identify 17 self-peptides recognized by neonatal tTreg cells, and reveal ligand specificity patterns that include self-antigens presented in an age- and inflammation-dependent manner. Fate-mapping studies of neonatal peptidyl arginine deiminase type IV (Padi4)-specific thymocytes reveal disparate fate choices. Neonatal thymocytes expressing T cell receptors that engage IAb-Padi4 with moderate dwell times within a conventional docking orientation are exported as tTreg cells. In contrast, Padi4-specific T cell receptors with short dwell times are expressed on CD4+ T cells, while long dwell times induce negative selection. Temporally, Padi4-specific thymocytes are subject to a developmental stage-specific change in negative selection, which precludes tTreg cell development. Thus, a temporal switch in negative selection and ligand binding kinetics constrains the neonatal tTreg selection window.

ISWI ATPase Smarca5 Regulates Differentiation of Thymocytes Undergoing β-Selection.

Development of lymphoid progenitors requires a coordinated regulation of gene expression, DNA replication, and gene rearrangement. Chromatin-remodeling activities directed by SWI/SNF2 superfamily complexes play important roles in these processes. In this study, we used a conditional knockout mouse model to investigate the role of Smarca5, a member of the ISWI subfamily of such complexes, in early lymphocyte development. Smarca5 deficiency results in a developmental block at the DN3 stage of αβ thymocytes and pro-B stage of early B cells at which the rearrangement of Ag receptor loci occurs. It also disturbs the development of committed (CD73+) γδ thymocytes. The αβ thymocyte block is accompanied by massive apoptotic depletion of β-selected double-negative DN3 cells and premitotic arrest of CD4/CD8 double-positive cells. Although Smarca5-deficient αβ T cell precursors that survived apoptosis were able to undergo a successful TCRβ rearrangement, they exhibited a highly abnormal mRNA profile, including the persistent expression of CD44 and CD25 markers characteristic of immature cells. We also observed that the p53 pathway became activated in these cells and that a deficiency of p53 partially rescued the defect in thymus cellularity (in contrast to early B cells) of Smarca5-deficient mice. However, the activation of p53 was not primarily responsible for the thymocyte developmental defects observed in the Smarca5 mutants. Our results indicate that Smarca5 plays a key role in the development of thymocytes undergoing β-selection, γδ thymocytes, and also B cell progenitors by regulating the transcription of early differentiation programs.

PF460 SINGLE CELL RNASEQ REVEALS THE IN VIVO DIFFERENTIATION POTENTIAL AND TRANSCRIPTIONAL DYNAMICS OF IMMATURE HUMAN THYMOCYTES

Background:T cell development depends on the colonization of the thymus by multipotent progenitor cells from the bone marrow. There, these precursor cells are gradually reprogrammed into committed T cell progenitors that have lost the developmental potential towards other hematopoietic lineages. While lineage tracing experiments in mice have provided evidence to which non T‐cell lineages these early T cell precursors differentiate in vivo, it remains unclear in human which blood cell lineages can originate from early T cell precursors. In vitro differentiation experiments have revealed erythroid, myeloid (DC, monocyte, granulocyte) and lymphoid (B, T, NK) differentiation potential, but whether this occurs in vivo is not clear.Aims:While lineage tracing experiments in mice have provided evidence to which non T‐cell lineages these early T cell precursors differentiate in vivo, it remains unclear in human which blood cell lineages can originate from early T cell precursors. In vitro differentiation experiments have revealed erythroid, myeloid (DC, monocyte, granulocyte) and lymphoid (B, T, NK) differentiation potential, but whether this occurs in vivo is not clear.Methods:Therefore, we have performed single cell RNA sequencing on more than 70.000 human CD34+lin‐ thymocytes that comprise the most immature thymocyte fraction of the human pediatric thymus.Results:Analysis of this dataset showed that cell cycle constitutes the majority of variation in the dataset, indicating less heterogeneity than anticipated. Indeed, using a strategy in which both clustering and trajectory inference were used, only limited branching was observed towards other, non T‐cell hematopoietic lineages, indicating a discrepancy between reported developmental potential and in vivo cell fate. Using these branching events, commitment was traced to coincide with loss of CD44 expression, confirming recent findings. In addition, we used CITEseq to correlate previously defined immature thymocyte subsets to these newly defined developmental cell states.Summary/Conclusion:In conclusion, our work suggests that the in vivo differentiation of immature human thymocytes is much more restricted compared to what in vitro differentiation experiments have suggested. In addition, we have delineated the transcriptional events that define the transition from multipotency toward T‐cell lineage commitment at the single cell level and redefine immature thymocyte subsets in human.

Long-term survival and differentiation of human thymocytes in human thymus-grafted immunodeficient mice.

Aim: Thymus transplants have produced encouraging clinical outcomes in achieving thymopoiesis and T-cell development. This study was aimed to investigate whether human thymus contains self-renewing lymphoid progenitors capable of maintaining long-term T-cell development. Materials & methods: Immunodeficient mice were transplanted with human thymic tissue along with autologous GFP-expressing or allogeneic CD34+ cells and followed for human thymopoiesis and T-cell development from the thymic progenitors versus CD34+ cells, which can be distinguished by GFP or HLA expression. Results: In both models, long-term thymopoiesis and T-cell development from the thymic grafts were detected. In these mice, human thymic progenitor-derived T cells including CD45RA+CD31+CD4+ new thymic emigrants were persistently present in the periphery throughout the observation period (32weeks). Conclusion: The results indicate that human thymus contains long-lived lymphoid progenitors that can maintain durable thymopoiesis and T-cell development.

Up-regulation of THY1 attenuates interstitial pulmonary fibrosis and promotes lung fibroblast apoptosis during acute interstitial pneumonia by blockade of the WNT signaling pathway

ABSTRACTAcute interstitial pneumonia (AIP) is an idiopathic pulmonary disease featuring rapid progressive dyspnea and respiratory failure. These symptoms typically develop within several days or weeks in patients without any pre-existing lung disease or external chest disease. Thymocyte differentiation antigen-1 (THY1) has been reported to have an effect on lung fibroblast proliferation and fibrogenic signaling. In this study, the mechanism of THY1 in AIP in influencing pulmonary fibrosis in terms of lung fibroblast proliferation and apoptosis was examined. An AIP mouse model with the pathological changes of lung tissues observed was established to identify the role of THY1 in the pathogenesis of AIP. The expression of THY1, a key regulator of the WNT pathway β-catenin and fibroblasts markers MMP-2, Occludin, α-SMA and Vimentin were determined. Lung fibroblasts of mice were isolated, in which THY1 expression was altered to identify roles THY1 plays in cell viability and apoptosis. A TOP/TOPflash assay was utilized to determine the activation of WNT pathway. Decrement of pulmonary fibrosis was achieved through THY1 up-regulation. The expression of MMP-2, Occludin, α-SMA, Vimentin and β-catenin, and the extent of β-catenin phosphorylation, significantly decreased, thereby indicating that THY1 overexpression inactivated WNT. Cell proliferation was inhibited and apoptosis was accelerated in lung fibroblasts transfected with vector carrying overexpressed THY1. Altogether, this study defines the potential role of THY1 in remission of AIP, via the upregulation of THY1, which renders the WNT pathway inactive. This inactivation of the WNT signaling pathway could alleviate pulmonary fibrosis by reducing lung fibroblast proliferation in AIP.Abbreviations: AIP: Acute interstitial pneumonia; ILDs: interstitial lung diseases; DAD: diffuse alveolar damage; SPF: specific-pathogen-free; NC: negative control; HCMV: human cytomegalovirus; HE: Hematoxylin-eosin; RIPA: radio-immunoprecipitation assay; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA: bovine serum albumin; HRP: horseradish peroxidase; ECL: electrochemiluminescence; FBS: fetal bovine serum; DMSO: dimethyl sulfoxide; OD: optical density

Abstract B198: Dissection of RORγt functions in lymphoid cell fate determination and differentiation

Abstract RORγt plays vital roles in development and differentiation of multiple immune cell types with pleiotropic functions, including thymocytes, LTi cells, ILC3, γδ T-cells and Th17 cells. Moreover, RORγ, the isoform of RORγt, was reported to promote cancer cell survival in castration-resistant prostate cancer. RORγ is expressed in tissues like muscle, liver and brain, but its function remains unclear. To address the mechanisms by which RORγt/RORγ contributes to diverse cell fates and differentiation programs, we are exploring RORγt post-translational modifications, RORγt/RORγ-associated protein complexes and downstream signals. To identify RORγt/RORγ-associated protein complexes, we generated knock-in mice expressing Strep-tagged RORγt/RORγ, which had IL-17A inducing activity similar to that of non-tagged RORγt in Th17 cells polarized in vitro and allowed for highly specific isolation of protein complexes. Strep-tagged RORγt proteins will be precipitated from thymocytes and polarized pathogenic or nonpathogenic Th17 cells, followed by mass-spectrometry to identify RORγt-associated protein partners and analysis of their functions in RORγt expressing cells. Moreover, due to its nonoverlapping expression patterns with RORγt and obscure functions, RORγ is very interesting to investigate in terms of associated protein complexes and roles in other tissues. RORγt mutations were employed to dissect its roles in cell fate determination and differentiation. We identified a RORγt mutant by random mutagenesis, designated as MUT, which dramatically lost transcriptional activity when expressed in 293T-cells and in vitro polarized Rorc-KO Th17 cells. Mice harboring MUT mutation had normal thymocyte differentiation and development of secondary lymphoid tissues. No lymphomas were detected in MUT mice, unlike in C57BL/6 RORγt-KO mice that develop T-cell lymphomas. Strikingly, T-cells and ILCs differed in proportions and numbers according to tissues. In secondary lymphoid organs, including draining lymph nodes and spleen, no differences were detected in proportions or numbers of CD4 or CD8 T-cells. In large intestine, however, the proportions and numbers of γδ T-cells and Th17 cells were significantly reduced in MUT mice, while those of Th2 cells were markedly increased. Meanwhile, hyper-proliferation of RORγt-negative T helper cells and Treg cells was detected in large intestine of MUT mice. ILCs from large intestine, including ILC3 with high expression of RORγt, were similar to wild type mice in both proportions and numbers. A different phenotype was detected in small intestine, where T-cells and ILCs were markedly reduced. Given the different requirement or expression stage of RORγt for development of T-cells and ILCs, the results probably reflect the effects of RORγt-associated functions on microenvironments. Analyses of immune cells and microbiome at different ages may help to address the mechanism. The functions of MUT RORγt-expressing immune cells will be explored in homeostatic or pathogenic settings with SFB colonization or the EAE model. Moreover, comparison of transcriptomes of wild-type and MUT RORγt expressing cells may reveal essential targets for differentiation and function, which will be validated by transfer of bone marrow or T-cells engineered by CRISPR-mediated gene knockout or retroviral overexpression. Wild-type and MUT RORγt-associated protein complexes will be compared to interrogate potential mechanisms in directing cell differentiation. Detected by mass-spectrometry and biochemical technologies, the majority of RORγt proteins are phosphorylated in naïve thymocytes and in vitro polarized Th17 cells. Mice with respective mutations in RORγt were generated by CRISPR technology. Similar analysis as indicated above will be performed to interrogate roles of RORγt in cell fate determination and differentiation. Citation Format: Hao Xu, Dan Littman, Wendy Huang. Dissection of RORγt functions in lymphoid cell fate determination and differentiation [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B198.

Targeted deletion of the aryl hydrocarbon receptor in dendritic cells prevents thymic atrophy in response to dioxin.

In nearly every species examined, administration of the persistent environmental pollutant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD) causes profound immune suppression and thymic atrophy in an aryl hydrocarbon receptor (AhR) dependent manner. Moreover, TCDD alters the development and differentiation of thymocytes, resulting in decreases in the relative proportion and absolute number of double positive (DP, CD4+CD8+) thymocytes, as well as a relative enrichment in the relative proportion and absolute number of double negative (DN, CD4-CD8-) and single-positive (SP) CD4+CD8- and CD4-CD8+ thymocytes. Previous studies suggested that the target for TCDD-induced thymic atrophy resides within the hemopoietic compartment and implicated apoptosis, proliferation arrest of thymic progenitors, and emigration of DN thymocytes to the periphery as potential contributors to TCDD-induced thymic atrophy. However, the precise cellular and molecular mechanisms involved remain largely unknown. Our results show that administration of 10 µg/kg TCDD and 8mg/kg 2-(1H-indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE) induced AhR-dependent thymic atrophy in mice on day 7, whereas 100mg/kg indole 3-carbinol (I3C) did not. Though our studies demonstrate that TCDD triggers a twofold increase in the frequency of apoptotic thymocytes, TCDD-induced thymic atrophy is not dependent on Fas-FasL interactions, and thus, enhanced apoptosis is unlikely to be a major mechanistic contributor. Finally, our results show that activation of the AhR in CD11c+ dendritic cells is directly responsible for TCDD-induced alterations in the development and differentiation of thymocytes, which results in thymic atrophy. Collectively, these results suggest that CD11c+ dendritic cells play a critical role in mediating TCDD-induced thymic atrophy and disruption of T lymphocyte development and differentiation in the thymus.

Lipopolysaccharide regulates the expression of thymocyte differentiation antigen-1 and integrin β3 of lung fibroblasts and inhibit autophagy

Objective To clarify the effect of lipopolysaccharide (LPS) on the expression of thymocyte differentiation antigen-1(Thy-1) and integrin β3 in mouse lung fibroblasts and its relationship with autophagy inhibition. Methods Primary cultured mouse lung fibroblasts were divided into four groups (n=3) based on different concentration of LPS stimulation: PBS control group (Con group), 0.25 mg/L LPS group (LPS0.25 group), 0.50 mg/L LPS group (LPS0.50 group), 1.00 mg/L LPS group (LPS1.00 group). Then the total cellular proteins were extracted. The expression of Thy-1 and integrin β3 were detected by Western blot. The expression of autophagy-related proteins LC3-Ⅱ/Ⅰ and Beclin-1 in cells was compared at 30 h after LPS stimulatin. The formation of autophagic bodies was observed by using transmission electron microscopy. Results Thy-1 protein expression was decreased in lung fibroblasts with increasing concentration of LPS stimulation. Compared with the control group(P<0.05), the level of Thy-1 protein in lung fibroblasts was significantly decreased in 1.00 mg/L LPS group. Integrin β3 protein expression was increased with increasing concentration of LPS stimulation. As a result, the expression leve of integrin in 1.00 mg/L LPS group was significantly higher than the level in control group (P<0.05). At the same time, LPS significantly inhibited the expression of autophagy-related proteins LC3-Ⅱ/Ⅰ(P<0.05) and Beclin-1 (P<0.05) in mouse lung fibroblasts and the formation of autophagic bodies(P<0.05). Conclusions LPS can down-regulate the expression of Thy-1 but up-regulate the expression of integrin β3. It also inhibits the expression of autophagy-related proteins LC3-Ⅱ/Ⅰ, Beclin-1 and the formation of autophagic bodies. This process may be associated with LPS-induced autophagy inhibition of lung fibroblasts and the progress of lung fibrosis. Key words: Lipopolysaccharide; Lung fibroblasts; Autophagy; Thymocyte differentiation antigen-1; Integrin β3

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy.

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes´ dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Polycomb Repressive Complex 2 is essential for development and maintenance of a functional TEC compartment

Thymic epithelial cells (TEC) are essential for thymocyte differentiation and repertoire selection. Despite their indispensable role in generating functional T cells, the molecular mechanisms that orchestrate TEC development from endodermal progenitors in the third pharyngeal pouch (3rd PP) are not fully understood. We recently reported that the T-box transcription factor TBX1 negatively regulates TEC development. Although initially expressed throughout the 3rd PP, Tbx1 becomes downregulated in thymus-fated progenitors and when ectopically expressed impairs TEC progenitor proliferation and differentiation. Here we show that ectopic Tbx1 expression in thymus fated endoderm increases expression of Polycomb repressive complex 2 (PRC2) target genes in TEC. PRC2 is an epigenetic modifier that represses gene expression by catalyzing trimethylation of lysine 27 on histone H3. The increased expression of PRC2 target genes suggests that ectopic Tbx1 interferes with PRC2 activity and implicates PRC2 as an important regulator of TEC development. To test this hypothesis, we used Foxn1Cre to delete Eed, a PRC2 component required for complex stability and function in thymus fated 3rd PP endoderm. Proliferation and differentiation of fetal and newborn TEC were disrupted in the conditional knockout (EedCKO) mutants leading to severely dysplastic adult thymi. Consistent with PRC2-mediated transcriptional silencing, the majority of differentially expressed genes (DEG) were upregulated in EedCKO TEC. Moreover, a high frequency of EedCKO DEG overlapped with DEG in TEC that ectopically expressed Tbx1. These findings demonstrate that PRC2 plays a critical role in TEC development and suggest that Tbx1 expression must be downregulated in thymus fated 3rd PP endoderm to ensure optimal PRC2 function.