Rhythm and Refrain: In Between Philosophy and Arts (2016)

<p>Pictorial representation of few important host defense response and apoptosis related genes that were differentially expressed (up regulated [green]; down regulated [red]) in HEV replicon transfected cell cultures compared to pcDNA3 only control.</p

Motifs in the cytoplasmic tail of the M-4 isoform that regulate cell surface expression.

<p>(<b>A</b>) Potential amino acid motifs in the cytoplasmic tail of M-4 isoform (<a href="http://www.expasy.org" target="_blank">www.expasy.org</a>). (<b>B</b>) Quantitative analysis of the surface expression of the M-4 wild-type and mutant proteins expressed in H9 cell line using the anti-CD8β antibody (5F2) by flow cytometry. The amount of CD8β binding was normalized to GFP expression. Each value corresponds to an average of three experiments. The standard deviation and two-population Student’s paired t-test was used to determine statistical differences of the mutants relative to the wild type M-4 isoform, indicated as one star * for p<0.05 and ** p<0.01. (<b>C</b>) Surface expression levels of M-4 wild-type and mutant proteins normalized to GFP expression in primary CD4⁺ T cells. Peripheral blood CD4⁺ T cells were stimulated with antibodies against CD3 and CD28 and transduced with lentiviruses expressing GFP and wild type or mutant M-4 proteins. On day 8 cell surface staining with CD8β antibody was analyzed by flow cytometry. The data are the mean +/− S.D. from three independent experiments. Values that are statistically different from the wild type M-4 protein are indicated as * p<0.05 and ** for p<0.01. (<b>D</b>) CD8αβ expression on CD4⁺ T cells prepared as in (C) expressing M-4 wild-type or mutants S²³²A or LL^235–6AG/IL^240–1AA. One representative experiment of five experiments is shown.</p

Differentially expressed genes in HEV, HBV and HEV+HBV transfected cell cultures compared to pcDNA3 vector-only control.

<p>The differential gene expression by Anova for each comparison, HEV vs. pcDNA3, HBV vs. pcDNA3 and HBV+HEV vs. pcDNA3 resulted in (A) 306, (B) 408 and (C) 461 genes with more than +/−2 fold change in expression (up regulated genes are marked in blue and down regulated genes are marked in red) and p value <0.05, respectively. (D) The Venn diagram shows common genes in the three groups.</p

The NPW motif in the M-4 cytoplasmic tail mediates binding to ubiquitinated proteins.

<p>(<b>A</b>) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, and CD8β wild type M1, M4 or chimeric protein M1 with 15 amino acids of the C terminus of the M4 cytoplasmic tail. After 48 hrs cells were lysed with 1% BRIJ 97, precipitated with anti-CD8β mAb and run on a polyacrylamide gel. Western blotting was performed with the indicated antibody. The membrane was then stripped and re-probed with the anti-CD8β antibody. The experiment was repeated three times. (<b>B</b>) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, and CD8β wild type or mutant proteins. The immunoprecipitation and Western blotting experiments were performed as in (A). A representative of four experiments is depicted. (<b>C</b>) The intensity of the 30 kDa band was analyzed and the amount of the 30 kDa band of the wild type relative to each mutant protein is represented. The levels of CD8β protein detected after reprobing with anti-CD8β antibody were used to correct for differences in protein expression between experiments. Student’s paired t-test was used to determine statistical differences of the mutants relative to the wild type M-4 isoform, indicated as one star * for p<0.05.</p

The Human CD8β M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

<div><p>The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.</p> </div

ERCC spike-in mixes QAQC.

<p>ERCC spike-in mixes QAQC.</p

CD4⁺ T cells transduced with the M-4 CD8β isoform showed increased frequency of cells producing MIP-1β after stimulation.

<p>Peripheral blood CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 24 hours, and then co-transduced with a lentivirus expressing a NY-ESO-1 TCR and another lentivirus expressing CD8α and one of the CD8β isoforms. Cells were stimulated and analyzed for cytokine/chemokine production after day 10–12. (<b>A</b>) Schematic representation of lentiviral vectors used for co-transduction of primary CD4⁺ T cells is followed by histograms for surface expression of CD8αβ, CD8α and TCR and dot plots showing cell population co-expressing NY-ESO-1 TCR and CD8α. Data were collected by flow cytometry using antibodies against CD8 and an MHC tetramer specific to NY-ESO-1 (NY-ESO- tetramer). Live CD3⁺ lymphocytes were gated using side vs. forward scatter, anti-CD3 antibody and live/dead cell dye. (<b>B</b>) Frequency of transduced CD4⁺ T cells producing MIP-1β (top panel) after stimulation with K562 target cells expressing the NY-ESO-1 antigen. T cells without targets served as negative control and cells stimulated with PMA and Ionomycin (bottom panel) were used as positive control. One representative of three independent experiments is shown. Values that are statistically different from the wild type M-1 protein as determined by two-population Student’s paired t-test are indicated as one star (*) for p<0.05.</p

Indirect immunofluorescence for detection of HBV and HEV in dual transfected Huh-7 cell cultures.

<p>HBV (I to L), HEV (E to H) and HBV+HEV (M to P) replicon transfected Huh-7 cells were stained with anti-HBsAg rabbit polyclonal and anti-pORF2 mouse monoclonal primary antibodies, followed by Alexa 546 conjugated goat anti-rabbit and Alexa 488 conjugated goat anti-mouse secondary antibodies in an indirect immunofluorescence assay. The nuclei were counter stained with DAPI. The composite image (P) showed both HBV and HEV positive cells as well as HBV-only and HEV-only cells. Composite images H and L show positivity for HEV and HBV, respectively. Mock-transfected control showed no staining either for HBV or HEV (A to D).</p

Graphical representation of RNA-Seq derived differential expression of genes that were selected for real-time PCR validation.

<p>XY bar charts represent genes selected for real-time PCR validation and their fold changes obtained in RNA-seq analysis as compared to pcDNA3 control in HEV (A), HBV (B) and HBV+HEV (C) transfected cultures. Each gene was represented by the same color in the three graphs.</p

Post-alignment QAQC using Bowtie2 of the unaligned reads from Tophat2.

<p>Post-alignment QAQC using Bowtie2 of the unaligned reads from Tophat2.</p