CD81 interacts with the T cell receptor to suppress signaling

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling

Dynamic Modulation of Thymic MicroRNAs in Response to Stress

thymocyte subsets. Several of the differentially regulated murine thymic miRs are also stress responsive in the heart, kidney, liver, brain, and/or spleen. The most dramatic thymic microRNA down modulated is miR-181d, exhibiting a 15-fold reduction following stress. This miR has both similar and distinct gene targets as miR-181a, another member of miR-181 family. Many of the differentially regulated microRNAs have known functions in thymopoiesis, indicating that their dysregulation will alter T cell repertoire selection and the formation of naïve T cells. This data has implications for clinical treatments involving anti-inflammatory steroids, ablation therapies, and provides mechanistic insights into the consequences of infections

Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency

Life-threatening pulmonary influenza can be caused by inborn errors of type I and III IFN immunity. We report a 5-yr-old child with severe pulmonary influenza at 2 yr. She is homozygous for a loss-of-function IRF9 allele. Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-α2b. The transcriptome induced by IFN-α2b in the patient's cells is much narrower than that of control cells; however, induction of a subset of IFN-stimulated gene transcripts remains detectable. In vitro, the patient's cells do not control three respiratory viruses, influenza A virus (IAV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). These phenotypes are rescued by wild-type IRF9, whereas silencing IRF9 expression in control cells increases viral replication. However, the child has controlled various common viruses in vivo, including respiratory viruses other than IAV. Our findings show that human IRF9- and ISGF3-dependent type I and III IFN responsive pathways are essential for controlling IAV

Identification of the interacting protein partners of the ThPOK transcription factor

T lymphocytes are an essential part of the adaptive immune system. In the course of T lymphocyte development, one crucial step is the decision to commit to one of two lineages: CD4 and CD8 single positive (SP) T cells. The cellular and molecular mechanisms underlying T cell lineage commitment has long been the subject of intense debate. ThPOK, T-helper-inducing POZ/Krüppel like factor, is a zinc finger transcription factor containing both a Krüppel-like zinc finger domain and a BTP/POZ domain, which has been linked to homodimerization and recruitment of other proteins. During the development of thymocytes, the ThPOK protein mediates the differentiation of MHC-II restricted thymocytes into the CD4 SP lineage, using its N-terminal BTB/POZ domain. We screened a human thymic cDNA library against the BTB/POZ domain of the ThPOK protein to identify its interacting protein partners by using a conventional yeast two hybrid system. We identified putative interacting proteins by performing DNA sequencing and bioinformatics analysis on relevant prey protein encoding plasmids. We re-confirmed these interactions in yeast cells by secondary yeast two hybrid screens. We confirmed the biological relevance of the identified interactions by performing co-immunoprecipitation experiments in transfected mammalian tissue culture cells. We demonstrated that seven proteins, named POMP, MEF2B, TCF7, ZNF384, DPP7, HINT2 and PARP12 can interact with the BTB/POZ domain of the ThPOK protein

A small non-interface surface epitope in human IL18 mediates the dynamics and self-assembly of IL18-IL18BP heterodimers

Interleukin 18 (IL18) is a pro-inflammatory cytokine that modulates innate and adaptive immune responses. IL18 activity is tightly controlled by the constitutively secreted IL18 binding protein (IL18BP). PDB structures of human IL18 showed that a short stretch of amino acids between 68 and 81 adopted a disordered conformation in all IL18-IL18BP complexes while adopting a 310 helical structure in other IL18 structures, including the receptor complexes. The C74 of human IL18, which was reported to form a novel intermolecular disulfide bond in the human tetrameric assembly, is also located in this short epitope. These observations reflected the importance of this short surface epitope for the structure and dynamics of the IL18-IL18BP heterodimers. We have analyzed all known IL18-IL18BP complexes in the PDB by all-atom MD simulations. The analysis also included two computed complex models adopting a helical structure for the surface epitope. Heterodimer simulations showed a stabilizing impact of the small surface region at the helical form by reducing flexibility of the complex backbone. Analysis of the symmetry-related human IL18-IL18BP tetramer showed that the unfolding of this small surface region also contributed to the IL18-IL18BP stability through a completely exposed C74 sidechain to form an intermolecular disulfide bond in the self-assembled human IL18-IL18BP dimer. Our findings showed how the conformation of the short IL18 epitope between amino acids 68 and 81 would affect IL18 activity by mediating the intermolecular interactions of IL18

Transgenic Expression of MicroRNA-181d Augments the Stress-Sensitivity of CD4⁺CD8⁺ Thymocytes

<div><p>Physiological stress resulting from infections, trauma, surgery, alcoholism, malnutrition, and/or pregnancy results in a substantial depletion of immature CD4⁺CD8⁺ thymocytes. We previously identified 18 distinct stress-responsive microRNAs (miRs) in the thymus upon systemic stress induced by lipopolysaccharide (LPS) or the synthetic glucocorticoid, dexamethasone (Dex). MiRs are short, non-coding RNAs that play critical roles in the immune system by targeting diverse mRNAs, suggesting that their modulation in the thymus in response to stress could impact thymopoiesis. MiR-181d is one such stress-responsive miR, exhibiting a 15-fold down-regulation in expression. We utilized both transgenic and gene-targeting approaches to study the impact of miR-181d on thymopoiesis under normal and stress conditions. The over-expression of miR-181d in developing thymocytes reduced the total number of immature CD4⁺CD8⁺ thymocytes. LPS or Dex injections caused a 4-fold greater loss of these cells when compared with the wild type controls. A knockout mouse was developed to selectively eliminate miR-181d, leaving the closely spaced and contiguous family member miR-181c intact. The targeted elimination of just miR-181d resulted in a thymus stress-responsiveness similar to wild-type mice. These experiments suggest that one or more of three other miR-181 family members have overlapping or compensatory functions. Gene expression comparisons of thymocytes from the wild type versus transgenic mice indicated that miR-181d targets a number of stress, metabolic, and signaling pathways. These findings demonstrate that selected miRs enhance stress-mediated thymic involution <i>in vivo</i>.</p></div

MiR-181d transgenic mice.

<p>(A) Schematic shows the sequence homology between mature miR-181 family members. 5′-seed region is underlined. Base differences are shaded with gray. (B) MiR-181d expression in various tissues examined by Northern blotting. U6 probe was used as the endogenous control. (C) Cloning of the pri-miR-181d into the VA-hCD2 transgenic cassette. Stem-loop structure of pre-miR-181d is shown, in which mature miR-181d is highlighted in blue. (D) Relative miR-181d levels were determined by real-time quantitative PCR. Littermate control values were set to 1. Graph represents the mean fold changes +/− SEM normalized to the U6 levels from 3 independent samples, performed in triplicates (n.s.  =  non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Two-tailed unpaired Student's <i>t</i>-test).</p

MiR-181d over-expression reduces the number of DP thymocytes.

<p>(A) Total thymus cellularity in the control and miR-181d Tg mice. (B) Representative plots show CD4 by CD8 profiles of thymocytes in the control and miR-181d Tg mice, analyzed by FACS. (C) Average percentages of thymocyte subsets (DN, DP, CD4 SP, and CD8 SP) from the control and miR-181d Tg mice. (D) Absolute cell numbers of DP thymocytes. (E) Absolute cell numbers of CD4 SP (left) and CD8 SP (right) thymocytes. (A–E) Data are from WT (n = 18), Tg-8 (n = 25), and Tg-38 (n = 16) mice. (F) Total thymus cellularity of the OTII Tg and OTII/miR-181d Tg-38 mice. (G) Total thymocytes were stained for CD4 and CD8, and analyzed by FACS. (H) Average percentages of DP and CD4 SP thymocytes are shown. (I) Histogram shows the surface expression of TCR (TCR Vα2) gated on CD4⁺CD8⁻ SP thymocytes from the OTII Tg (dark gray) and OTII/miR-181d Tg-38 mice (black line). (F–I) Data are from at least 2 mice per group. Each bar is the mean +/− SEM (n.s.  =  non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Two-tailed unpaired Student's <i>t</i>-test). (J) Histograms show CD69 expression on CD4 SP and CD8 SP thymocytes from the WT (white), Tg-8 (light gray), and Tg-38 (dark gray) mice. (K) Relative MFI (Mean Fluorescence Intensity) levels of CD69 on SP thymocytes. (L) Ratio of the CD69⁺TCRβ^high to CD69⁻TCRβ^high thymocyte numbers shown for CD4 SP and CD8 SP thymocytes. (M) Average percentages of Annexin V⁺ cells gated on DP thymocytes. (J-M) Data are of at least 3 mice per group. All bar graphs represent the mean +/− SEM values (n.s.  =  non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; One-way ANOVA followed by Tukey's post-hoc test).</p

Characterization of peripheral lymphocytes in miR-181d transgenic mice.

<p>(A) Total cellularity in the lymph nodes of the control and miR-181d Tg mice. (B) Representative FACS plots of CD4⁺ and CD8⁺ T cells in the lymph nodes. (C–D) Average percentages (C) and absolute numbers (D) of CD4⁺ and CD8⁺ T cells in the lymph nodes. (A–D) Data are of the mean +/− SEM from the WT (n = 17), Tg-8 (n = 23), and Tg-38 (n = 14) mice (n.s.  =  non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; One-way ANOVA followed by Tukey's post-hoc test). (E) CD4 and CD8 profiles of peripheral T cells from the lymph nodes of the OTII Tg and OTII/miR-181d Tg-38 mice. (F) Bar graph shows average percentages of CD4⁺ T lymphocytes in the lymph nodes. (G) Surface expression of TCR (TCR Vα2) gated on CD4⁺ T cells in the lymph nodes of the OTII Tg (dark gray) and OTII/miR-181d Tg-38 mice (black line). (E–G) Data are generated from at least 2 mice per group. Each bar represents the mean +/− SEM values (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Two-tailed unpaired Student's <i>t</i>-test).</p

MiR-181d over-expression elevates stress-induced thymic atrophy.

<p>(A) Representative plots show CD4 by CD8 profiles of total thymocytes from the control and miR-181d Tg mice at 72 hours after PBS or LPS (100 µg/mouse) injections. (B–C) Graphs demonstrate the average percentages of DP thymocytes (B), and CD4 SP and CD8 SP thymocytes (C) at 72 hours post-injection (PBS, white; LPS, black). (B–C) Data are of the mean +/− SEM from at least 4 independent experiments using at least 3 mice per injection (n.s.  =  non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Two-way ANOVA followed by Bonferroni's post-hoc test). (D–E) Data were calculated from the experiments shown in the panels A and B. Each bar shows the mean +/− SEM. (D) Ratios of DP thymocyte numbers upon LPS treatment to the numbers of DP thymocytes upon PBS treatment (n.s.  =  non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; One-way ANOVA followed by Tukey's post-hoc test). (E) Average percentages of Annexin V⁺ cells gated on DP thymocytes at 72 hours post-injection (PBS, white; LPS, black). (n.s.  =  non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Two-way ANOVA followed by Bonferroni's post-hoc test). (F) Total thymic cellularity in the control and miR-181d Tg-38 mice at 48 hours upon Dex injection (60 µg/mouse). (G) Representative FACS plots show CD4 by CD8 profiles of thymocytes after 48 hours post-Dex injection. (H–I) Average percentages (H) and absolute numbers (I) of thymocyte subsets following Dex treatment at 48 hours. (F–I) Bar graphs show the mean +/− SEM from at least 4 mice per treatment (n.s.  =  non-significant, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Two-tailed unpaired Student's <i>t</i>-test).</p