Knockout of SLy1 decreases double-negative thymocyte proliferation and protects mice from p53-induced tumor formation.

The lymphocyte-specific adapter protein SLy1 has previously been identified as indispensable for thymocyte development and T-cell proliferation and, recently, as a cause of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in SLy1KO and SLy1d/d mice. As SLy1KO NK cells show increased levels of p53, we focused our research on the interdependency of SLy1 and p53 for thymocyte development. Using RT-PCR and immunoblot analysis, we observed increased levels of p53 as well as DNA damage response proteins in SLy1KO thymocytes. To test for rescue from SLy1-induced deficiencies in thymocyte development like reduced thymocyte numbers and reduced DN to DP progression, we generated a mouse model with T cell-specific p53-deficiency on an SLy1KO background and analyzed lymphocyte populations in these mice and respective controls. Astonishingly, SLy1KO -typical deficiencies were retained, showing that SLy1 is mechanistically independent of p53. Studies of apoptosis and proliferation in SLy1KO thymocytes revealed decreased proliferation in the DN3 subpopulation as a possible reason for the decreased thymocyte number. In mice with p53-deficient T cells, we observed tumor formation leading to reduced survival, preferentially in SLy1WT mice. Thus, we suggest that a SLy1-deficiency reduces proliferation, resulting in less hematologic tumors initiated by the p53-deficiency.

ICAP-1 loss impairs CD8+ thymocyte development and leads to reduced marginal zone B cells in mice.

ICAP-1 regulates ß1-integrin activation and cell adhesion. Here, we used ICAP-1-null mice to study ICAP-1 potential involvement during immune cell development and function. Integrin α4ß1-dependent adhesion was comparable between ICAP-1-null and control thymocytes, but lack of ICAP-1 caused a defective single-positive (SP) CD8+ cell generation, thus, unveiling an ICAP-1 involvement in SP thymocyte development. ICAP-1 bears a nuclear localization signal and we found it displayed a strong nuclear distribution in thymocytes. Interestingly, there was a direct correlation between the lack of ICAP-1 and reduced levels in SP CD8+ thymocytes of Runx3, a transcription factor required for CD8+ thymocyte generation. In the spleen, ICAP-1 was found evenly distributed between cytoplasm and nuclear fractions, and ICAP-1-/- spleen T and B cells displayed upregulation of α4ß1-mediated adhesion, indicating that ICAP-1 negatively controls their attachment. Furthermore, CD3+ - and CD19+ -selected spleen cells from ICAP-1-null mice showed reduced proliferation in response to T- and B-cell stimuli, respectively. Finally, loss of ICAP-1 caused a remarkable decrease in marginal zone B- cell frequencies and a moderate increase in follicular B cells. Together, these data unravel an ICAP-1 involvement in the generation of SP CD8+ thymocytes and in the control of marginal zone B-cell numbers.

A general chemical crosslinking strategy for structural analyses of weakly interacting proteins applied to preTCR-pMHC complexes.

T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αßT lymphocytes through their ß chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide-MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αßTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRß-pMHC interaction. The linkage model of preTCRß-pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR-pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.

MicroRNA-142 controls thymocyte proliferation.

T-cell development is a spatially and temporally regulated process, orchestrated by well-defined contributions of transcription factors and cytokines. Here, we identify the noncoding RNA miR-142 as an additional regulatory layer within murine thymocyte development and proliferation. MiR-142 deficiency impairs the expression of cell cycle-promoting genes in mature mouse thymocytes and early progenitors, accompanied with increased levels of cyclin-dependent kinase inhibitor 1B (Cdkn1b, also known as p27Kip1 ). By using CRISPR/Cas9 technology to delete the miR-142-3p recognition element in the 3'UTR of cdkn1b, we confirm that this gene is a novel target of miR-142-3p in vivo. Increased Cdkn1b protein expression alone however was insufficient to cause proliferation defects in thymocytes, indicating the existence of additional critical miR-142 targets. Collectively, we establish a key role for miR-142 in the control of early and mature thymocyte proliferation, demonstrating the multifaceted role of a single miRNA on several target genes.

Recirculating Th2 cells induce severe thymic dysfunction via IL-4/STAT6 signaling pathway.

Thymic involution happened early in life, but a certain ratio of activated CD4+ T cells will persistently recirculate into the thymus from the periphery and it have been suggested to be able to inhibit the development of embryonic thymocytes. Our present study was aimed to elucidate the specific mechanism how activated CD4+ T cells could influence upon developing thymocytes by using fetal thymic organ culture (FTOC) and kidney capsule transplantation. Our results demonstrated that Th2 cells were found to play a fundamental role in the inhibition of embryonic thymocyte development since a very low concentration of Th2 cells could obviously reduce the total number of thymocytes. And this effect was not tenable in other Th cell type. Notably, IL-4, the major cytokine secreted by Th2 cells, was suggested the key factor playing the inhibition role. In addition to reduced cell population, the proportion of double positive (DP) T cells was also heavily decreased. Furthermore, we demonstrated that it was the downstream effector signal transducer and activator of transcription 6 (STAT6) of IL-4 partially manipulate this inhibition. Together, these findings reveal a novel influence of Th2 cells re-entering the thymus on thymic involution.

Altered thymocyte and T cell development in neonatal mice with hyperoxia-induced lung injury.

BACKGROUND: The adaptive immune system of neonates is relatively underdeveloped. The thymus is an essential organ for adaptive T cell development and might be affected during the natural course of oxygen induced lung injury. The effect of prolonged hyperoxia on the thymus, thymocyte and T cell development, and its proliferation has not been studied extensively. METHODS: Neonatal mice were exposed to 85% oxygen (hyperoxia) or room air (normoxia) up to 28 days. Flow cytometry using surface markers were used to assay for thymocyte development and proliferation. RESULTS: Mice exposed to prolonged hyperoxia had evidence of lung injury associated alveolar simplification, a significantly lower mean weight, smaller thymic size, lower mean thymocyte count and higher percentage of apoptotic thymocytes. T cells subpopulation in the thymus showed a significant reduction in the count and proliferation of double positive and double negative T cells. There was a significant reduction in the count and proliferation of single positive CD4+ and CD8+ T cells. CONCLUSIONS: Prolonged hyperoxia in neonatal mice adversely affected thymic size, thymocyte count and altered the distribution of T cells sub-populations. These results are consistent with the hypothesis that prolonged hyperoxia causes defective development of T cells in the thymus.

Leucine-rich repeat containing 8A (LRRC8A)-dependent volume-regulated anion channel activity is dispensable for T-cell development and function.

BACKGROUND: Leucine-rich repeat containing 8A (LRRC8A) is an ubiquitously expressed transmembrane protein with 17 leucine-rich repeats (LRRs) at its C-terminal end and is an essential component of the volume-regulated anion channel (VRAC), which controls cellular volume. A heterozygous mutation in LRRC8A that truncates the 2 terminal LRRs was reported in a patient with agammaglobulinemia and absent B cells and was demonstrated to exert a dominant negative effect on T- and B-cell development in mice. Lrrc8a-/- mice have severely defective T-cell development and function. It is not known whether the T- and B-cell defects caused by LRRC8A deficiency are caused by loss of VRAC activity. OBJECTIVE: We sought to determine whether VRAC activity is required for normal T-cell development and function. METHODS: VRAC activity was examined by using patch-clamp analysis. Flow cytometry was used to examine T-cell development. T-cell proliferation, cytokine secretion, and antibody titers were measured by using standard techniques. RESULTS: We demonstrate that the spontaneous mouse mutant ébouriffé (ebo/ebo) harbors a homozygous 2-bp frameshift mutation in Lrrc8a that truncates the 15 terminal LRRs of LRRC8A. The Lrrc8aebo mutation does not affect protein expression but drastically diminishes VRAC activity in T cells. ebo/ebo mice share features with Lrrc8a-/- mice that include curly hair, infertility, reduced longevity, and kidney abnormalities. However, in contrast to Lrrc8a-/- mice, ebo/ebo mice have normal T-cell development and function and intact antibody response to T-dependent antigen. CONCLUSION: LRRC8A-dependent VRAC activity is dispensable for T-cell development and function.

Targeted loss of SHP1 in murine thymocytes dampens TCR signaling late in selection.

SHP1 is a tyrosine phosphatase critical to proximal regulation of TCR signaling. Here, analysis of CD4-Cre SHP1(fl/fl) conditional knockout thymocytes using CD53, TCRß, CD69, CD4, and CD8α expression demonstrates the importance of SHP1 in the survival of post selection (CD53(+) ), single-positive thymocytes. Using Ca(2+) flux to assess the intensity of TCR signaling demonstrated that SHP1 dampens the signal strength of these same mature, postselection thymocytes. Consistent with its dampening effect, TCR signal strength was also probed functionally using peptides that can mediate selection of the OT-I TCR, to reveal increased negative selection mediated by lower-affinity ligand in the absence of SHP1. Our data show that SHP1 is required for the survival of mature thymocytes and the generation of the functional T-cell repertoire, as its absence leads to a reduction in the numbers of CD4(+) and CD8(+) naïve T cells in the peripheral lymphoid compartments.

Dynamic control of ß1 integrin adhesion by the plexinD1-sema3E axis.

Plexins and semaphorins comprise a large family of receptor-ligand pairs controlling cell guidance in nervous, immune, and vascular systems. How plexin regulation of neurite outgrowth, lymphoid trafficking, and vascular endothelial cell branching is linked to integrin function, central to most directed movement, remains unclear. Here we show that on developing thymocytes, plexinD1 controls surface topology of nanometer-scaled ß1 integrin adhesion domains in cis, whereas its ligation by sema3E in trans regulates individual ß1 integrin catch bonds. Loss of plexinD1 expression reduces ß1 integrin clustering, thereby diminishing avidity, whereas sema3E ligation shortens individual integrin bond lifetimes under force to reduce stability. Consequently, both decreased expression of plexinD1 during developmental progression and a thymic medulla-emanating sema3E gradient enhance thymocyte movement toward the medulla, thus enforcing the orchestrated lymphoid trafficking required for effective immune repertoire selection. Our results demonstrate plexin-tunable molecular features of integrin adhesion with broad implications for many cellular processes.

Bcl11b prevents the intrathymic development of innate CD8 T cells in a cell intrinsic manner.

If Bcl11b activity is compromised, CD4(+)CD8(+) double-positive (DP) thymocytes produce a greatly increased fraction of innate CD8(+) single-positive (SP) cells highly producing IFN-γ, which are also increased in mice deficient of genes such as Itk, Id3 and NF-κB1 that affect TCR signaling. Of interest, the increase in the former two is due to the bystander effect of IL-4 that is secreted by promyelocytic leukemia zinc finger-expressing NKT and γδT cells whereas the increase in the latter is cell intrinsic. Bcl11b zinc-finger proteins play key roles in T cell development and T cell-mediated immune response likely through TCR signaling. We examined thymocytes at and after the DP stage in Bcl11b (F/S826G) CD4cre, Bcl11b (F/+) CD4cre and Bcl11b (+/S826G) mice, carrying the allele that substituted serine for glycine at the position of 826. Here we show that Bcl11b impairment leads to an increase in the population of TCRαß(high)CD44(high)CD122(high) innate CD8SP thymocytes, together with two different developmental abnormalities: impaired positive and negative selection accompanying a reduction in the number of CD8SP cells, and developmental arrest of NKT cells at multiple steps. The innate CD8SP thymocytes express Eomes and secrete IFN-γ after stimulation with PMA and ionomycin, and in this case their increase is not due to a bystander effect of IL-4 but cell intrinsic. Those results indicate that Bcl11b regulates development of different thymocyte subsets at multiple stages and prevents an excess of innate CD8SP thymocytes.

CD28 controls the development of innate-like CD8+ T cells by promoting the functional maturation of NKT cells.

NK T cells(NKT cells) share functional characteristics and homing properties that are distinct from conventional T cells. In this study, we investigated the contribution of CD28 in the functional development of γδ NKT and αß NKT cells in mice. We show that CD28 promotes the thymic maturation of promyelocytic leukemia zinc finger(+) IL-4(+) NKT cells and upregulation of LFA-1 expression on NKT cells. We demonstrate that the developmental defect of γδ NKT cells in CD28-deficient mice is cell autonomous. Moreover, we show in both wild-type C57BL/6 mice and in downstream of tyrosine kinase-1 transgenic mice, a mouse model with increased numbers of γδ NKT cells, that CD28-mediated regulation of thymic IL-4(+) NKT cells promotes the differentiation of eomesodermin(+) CD44(high) innate-like CD8(+) T cells. These findings reveal a previously unappreciated mechanism by which CD28 controls NKT-cell homeostasis and the size of the innate-like CD8(+) T-cell pool.

GRP75-dependent mitochondria-ER contacts ensure cell survival during early mouse thymocyte development.

Mitochondria and endoplasmic reticulum contacts (MERCs) control multiple cellular processes, including cell survival and differentiation. Based on the observations that MERCs were specifically enriched in the CD4-CD8- double-negative (DN) stage, we studied their role in early mouse thymocyte development. We found that T cell-specific knockout of Hspa9, which encodes GRP75, a protein that mediates MERC formation by assembling the IP3R-GRP75-VDAC complex, impaired DN3 thymocyte viability and resulted in thymocyte developmental arrest at the DN3-DN4 transition. Mechanistically, GRP75 deficiency induced mitochondrial stress, releasing mitochondrial DNA (mtDNA) into the cytosol and triggering the type I interferon (IFN-I) response. The IFN-I pathway contributed to both the impairment of cell survival and DN3-DN4 transition blockage, while increased lipid peroxidation (LPO) played a major role downstream of IFN-I. Thus, our study identifies the essential role of GRP75-dependent MERCs in early thymocyte development and the governing facts of cell survival and differentiation in the DN stage.

Critical roles of the miR-17∼92 family in thymocyte development, leukemogenesis, and autoimmunity.

Thymocyte development requires precise control of PI3K-Akt signaling to promote proliferation and prevent leukemia and autoimmune disorders. Here, we show that ablating individual clusters of the miR-17∼92 family has a negligible effect on thymocyte development, while deleting the entire family severely impairs thymocyte proliferation and reduces thymic cellularity, phenocopying genetic deletion of Dicer. Mechanistically, miR-17∼92 expression is induced by Myc-mediated pre-T cell receptor (TCR) signaling, and miR-17∼92 promotes thymocyte proliferation by suppressing the translation of Pten. Retroviral expression of miR-17∼92 restores the proliferation and differentiation of Myc-deficient thymocytes. Conversely, partial deletion of the miR-17∼92 family significantly delays Myc-driven leukemogenesis. Intriguingly, thymocyte-specific transgenic miR-17∼92 expression does not cause leukemia or lymphoma but instead aggravates skin inflammation, while ablation of the miR-17∼92 family ameliorates skin inflammation. This study reveals intricate roles of the miR-17∼92 family in balancing thymocyte development, leukemogenesis, and autoimmunity and identifies those microRNAs (miRNAs) as potential therapeutic targets for leukemia and autoimmune diseases.

Studying T Cell Development in Neonatal and Adult Thymic Slices.

T cell development occurs in the thymus and is coordinated temporally and spatially within the highly complex thymic microenvironment. Therefore, T cell selection and maturation events cannot be fully recapitulated using traditional two-dimensional tissue culture in vitro. The thymic slice system provides a highly versatile system for studying T cell development ex vivo while preserving three-dimensional thymic architecture. Using the thymic slice system, T cell selection and maturation events can be visualized by live imaging and quantified by flow cytometry. Here we describe the process for generating slices from neonatal and adult mice.

Intrathymic somatotropic circuitry: consequences upon thymus involution.

Growth hormone (GH) is a classic pituitary-derived hormone crucial to body growth and metabolism. In the pituitary gland, GH production is stimulated by GH-releasing hormone and inhibited by somatostatin. GH secretion can also be induced by other peptides, such as ghrelin, which interacts with receptors present in somatotropic cells. It is well established that GH acts directly on target cells or indirectly by stimulating the production of insulin-like growth factors (IGFs), particularly IGF-1. Notably, such somatotropic circuitry is also involved in the development and function of immune cells and organs, including the thymus. Interestingly, GH, IGF-1, ghrelin, and somatostatin are expressed in the thymus in the lymphoid and microenvironmental compartments, where they stimulate the secretion of soluble factors and extracellular matrix molecules involved in the general process of intrathymic T-cell development. Clinical trials in which GH was used to treat immunocompromised patients successfully recovered thymic function. Additionally, there is evidence that the reduction in the function of the somatotropic axis is associated with age-related thymus atrophy. Treatment with GH, IGF-1 or ghrelin can restore thymopoiesis of old animals, thus in keeping with a clinical study showing that treatment with GH, associated with metformin and dehydroepiandrosterone, could induce thymus regeneration in healthy aged individuals. In conclusion, the molecules of the somatotrophic axis can be envisioned as potential therapeutic targets for thymus regeneration in age-related or pathological thymus involution.

Single-cell detection of primary transcripts, their genomic loci and nuclear factors by 3D immuno-RNA/DNA FISH in T cells.

Over the past decades, it has become increasingly clear that higher order chromatin folding and organization within the nucleus is involved in the regulation of genome activity and serves as an additional epigenetic mechanism that modulates cellular functions and gene expression programs in diverse biological processes. In particular, dynamic allelic interactions and nuclear locations can be of functional importance during the process of lymphoid differentiation and the regulation of immune responses. Analyses of the proximity between chromatin and/or nuclear regions can be performed on populations of cells with high-throughput sequencing approaches such as chromatin conformation capture ("3C"-based) or DNA adenine methyltransferase identification (DamID) methods, or, in individual cells, by the simultaneous visualization of genomic loci, their primary transcripts and nuclear compartments within the 3-dimensional nuclear space using Fluorescence In Situ Hybridization (FISH) and immunostaining. Here, we present a detailed protocol to simultaneously detect nascent RNA transcripts (3D RNA FISH), their genomic loci (3D DNA FISH) and/or their chromosome territories (CT paint DNA FISH) combined with the antibody-based detection of various nuclear factors (immunofluorescence). We delineate the application and effectiveness of this robust and reproducible protocol in several murine T lymphocyte subtypes (from differentiating thymic T cells, to activated splenic and peripheral T cells) as well as other murine cells, including embryonic stem cells, B cells, megakaryocytes and macrophages.

Effects of sex steroids on thymic epithelium and thymocyte development.

Sex steroid hormones have major effects on the thymus. Age-related increases in androgens and estrogens and pregnancy-induced increases in progestins all cause dramatic thymic atrophy. Atrophy can also be induced by treatment with exogenous sex steroids and reversed by ablation of endogenous sex steroids. Although these observations are frequently touted as evidence of steroid lymphotoxicity, they are often driven by steroid signaling in thymic epithelial cells (TEC), which are highly steroid responsive. Here, we outline the effects of sex steroids on the thymus and T cell development. We focus on studies that have examined steroid signaling in vivo, aiming to emphasize the actions of endogenous steroids which, via TEC, have remarkable programming effects on the TCR repertoire. Due to the dramatic effects of steroids on TEC, especially thymic involution, the direct effects of sex steroid signaling in thymocytes are less well understood. We outline studies that could be important in addressing these possibilities, and highlight suggestive findings of sex steroid generation within the thymus itself.

3D Genome Organization as an Epigenetic Determinant of Transcription Regulation in T Cells.

In the heart of innate and adaptive immunity lies the proper spatiotemporal development of several immune cell lineages. Multiple studies have highlighted the necessity of epigenetic and transcriptional regulation in cell lineage specification. This mode of regulation is mediated by transcription factors and chromatin remodelers, controlling developmentally essential gene sets. The core of transcription and epigenetic regulation is formulated by different epigenetic modifications determining gene expression. Apart from "classic" epigenetic modifications, 3D chromatin architecture is also purported to exert fundamental roles in gene regulation. Chromatin conformation both facilitates cell-specific factor binding at specified regions and is in turn modified as such, acting synergistically. The interplay between global and tissue-specific protein factors dictates the epigenetic landscape of T and innate lymphoid cell (ILC) lineages. The expression of global genome organizers such as CTCF, YY1, and the cohesin complexes, closely cooperate with tissue-specific factors to exert cell type-specific gene regulation. Special AT-rich binding protein 1 (SATB1) is an important tissue-specific genome organizer and regulator controlling both long- and short-range chromatin interactions. Recent indications point to SATB1's cooperation with the aforementioned factors, linking global to tissue-specific gene regulation. Changes in 3D genome organization are of vital importance for proper cell development and function, while disruption of this mechanism can lead to severe immuno-developmental defects. Newly emerging data have inextricably linked chromatin architecture deregulation to tissue-specific pathophysiological phenotypes. The combination of these findings may shed light on the mechanisms behind pathological conditions.

CycleFlow simultaneously quantifies cell-cycle phase lengths and quiescence in vivo.

Populations of stem, progenitor, or cancer cells show proliferative heterogeneity in vivo, comprising proliferating and quiescent cells. Consistent quantification of the quiescent subpopulation and progression of the proliferating cells through the individual phases of the cell cycle has not been achieved. Here, we describe CycleFlow, a method that robustly infers this comprehensive information from standard pulse-chase experiments with thymidine analogs. Inference is based on a mathematical model of the cell cycle, with realistic waiting time distributions for the G1, S, and G2/M phases and a long-term quiescent G0 state. We validate CycleFlow with an exponentially growing cancer cell line in vitro. Applying it to T cell progenitors in steady state in vivo, we uncover strong proliferative heterogeneity, with a minority of CD4+CD8+ T cell progenitors cycling very rapidly and then entering quiescence. CycleFlow is suitable as a routine method for quantitative cell-cycle analysis.

Identification of Rare Thymic NKT Cell Precursors by Multiparameter Flow Cytometry.

Mouse invariant natural killer T (NKT) cells are a subset of T lymphocytes which have been shown to play a significant role in innate and adaptive immune responses. Features of innate responses are attributed to these cells because they can be stimulated simultaneously with the same ligand to produce quickly and in large amount cytokines without prior immunization. Because these characteristics could be exploited for clinical applications, NKT cells have attracted considerable interest. Many studies have investigated the molecular mechanisms through which they are selected and differentiate. These studies are based on developmental models that serve as a scaffold to understand the specific roles played by various factors and to identify checkpoints during cellular development. Analysis of NKT cell precursors at the HSAhigh stage, stage 0, can reveal potential selection defects, whereas analysis of NKT cells at the HSAlow stage can shed light on defects in the maturation/differentiation of the different NKT cell subsets (NKT1, 2, and 17). Unlike HSAlow NKT cell subsets, HSAhigh NKT cell precursors are not accurately identified by flow cytometry because of their extreme rarity. Here, we describe an NKT cell enrichment strategy to identify unambiguously NKT cell precursors at the HSAhigh stage that can be used to assess their distribution and characteristics by multicolor flow cytometry.