WT CD28 can adopt a stable conformation that would allow bivalent binding.

<p>MD simulations were run with WT CD28 starting from a rotated dimer orientation predicted from the simulations starting from the CTLA-4 dimer orientation. (<b>A</b>) The RMSD with respect to the initial conformation for three independent trajectories indicate that these dimer conformers are very stable. (<b>B</b>) When CD80 molecules were docked onto the conformations, there was no surface buried between the ligands, indicating that all conformations were bivalent. A representative bivalent conformation of CD28 (green) with docked CD80 molecules (cyan) is shown to illustrate the CD28 dimer (<b>C</b>) and in a rotated view to show the orientation of the docked CD80 molecules (<b>D</b>).</p

WT CD28 adopts a bivalent binding conformation when starting from the CTLA-4 dimer orientation.

<p>MD simulations were run with WT CD28 starting from a CTLA-4 dimer orientation. (<b>A</b>) The RMSD of three independent trajectories with respect to the initial conformation are shown over time. There is considerable rearrangement of the subunits at the beginning of the simulations, reflected by an increase in the RMSD. The conformational fluctuations stabilize after the first 100 ns of simulation, although the trajectories adopt different conformations. This instability was not inherent to this dimer orientation, as the RMSD of MD simulations of CTLA-4 remained below 4Å (data not shown). (<b>B</b>) CD80 molecules were docked onto the simulated CD28 dimers to obtain the corresponding CD28-CD80 complexes. The surface area buried between the docked ligands was calculated at various times along each of the three independent trajectories to estimate the fraction of bivalent-competent conformations; the average over all three trajectories was 79%. (<b>C</b>) A representative conformation of CD28 (green) showing docked CD80 ligands (cyan) illustrates the potential for bivalent ligand binding.</p

TCR signaling can enhance the avidity of CD28 ligand binding.

<p>(<b>A</b>) Images from a cell adhesion assay. Top, a resting T cell and CD80-coated bead brought into contact using micropipets. Middle, the same T cell 30s later after the cell and bead were separated. No adhesion was observed. Bottom, image of a T cell bound to an anti-CD3 coated bead, which is out of the plane of focus, but clearly distal to the site of interaction of the T cell with the CD80-bead. This image was taken shortly after the T cell was pulled away from the CD80-bead. In this case, the T cell-CD80-bead adhesion has caused the T cell to dislodge from the micropipet. (<b>B</b>) Cell frequency adhesion of in vitro primed and resting WT DO11.10 T cells to control beads (None) or beads coated with CD80 (CD80-Fc) in the absence (Unstim, empty bars) and after (Anti-CD3; filled bars) stimulation. No increase in adhesion to CD80 was detected when cells were stimulated with beads coated with anti-MHC class I (not shown). (<b>C</b> and <b>D</b>) WT and CD28-deficient T cells were primed with CD80/CD86-negative APC to reduce upregulation of CTLA-4. (<b>C</b>) Flow cytometry of CTLA-4 expression on WT (thin line) and CD28-deficient (thick line) T cells after in vitro priming. Isotype control (filled) and WT T cells primed in the presence of CD28 costimulation (dashed line) are included as negative and positive controls. (<b>D</b>) Cell frequency adhesion of WT and CD28-deficient (CD28KO) T cells that were primed with CD80/CD86-negative cells, to CD80-beads in the absence (Unstim) and after (Anti-CD3) stimulation. (<b>E</b>) Adhesion frequency of WT and CTLA-4-deficient (CTLA-4KO) T cells to control beads (None) or beads coated with increasing concentrations of CD80 in the absence (Unstim) and after (Anti-CD3) stimulation. Cell adhesion data are presented as mean ± SD of the adhesion frequency for individual cells (25 impingements each; n = 9–10, except for 62.5 ng/ml CD80-Fc in panel E, n = 6; p values for t tests between samples are shown; ns, not significant).</p

K118I/K120P CD28 is polarized toward CD80-positive cells in the absence of TCR signaling.

<p>(<b>A</b> and <b>B</b>) CD28-deficient, DO11.10 CD4 T cells were retrovirally transduced with WT or K118I/K120P CD28 fused to YFP, stained with anti-CD28 and analyzed by flow cytometry. Two color display (<b>A</b>) shows total transduced protein expression as detected by YFP (FL1, x-axis) and cell surface expression of CD28 as detected by anti-CD28 staining (FL3, y-axis). Single color display (<b>B</b>) shows relative cell surface expression of WT CD28 (thick line) and K118I/K120P (KK/IP) CD28 (thin line). (<b>C</b> and <b>D</b>) Representative images of WT and K118I/K120P CD28-YFP localization in cell:cell conjugates with CD80-negative (<b>C</b>) and CD80-positive (<b>D</b>) APC in the presence (+Ag) and absence (-Ag) of TCR signaling. DIC (digital image correlation) and fluorescent images are shown. (<b>E</b>) Individual conjugates were visually scored for CD28 polarization toward the interacting APC and the percentage of conjugates displaying polarized CD28 is shown (n = 25–28). (<b>F</b>) The efficiency of CD28 recruitment to CD80-positive APC was calculated by determining the ratio of YFP fluorescence within the T cell:APC contact site to the YFP fluorescence in the T cell plasma membrane distal to the contact site. Values for individual cells, population medians and statistical analysis (non-parametric Kruskal-Wallis ANOVA with Dunn’s multiple comparison) are shown (ns, not significant; * p<0.05; ***p<0.001).</p

K118I/K120P CD28 binds CD80 with high avidity in the absence of TCR signaling.

<p>Cell frequency adhesion of CD28-deficient, DO11.10 T cells retrovirally transduced with WT CD28-YFP or K118I/K120P CD28-YFP to control beads (None) or beads coated with CD80 in the absence (Unstim) and after (Anti-CD3) stimulation. Cell adhesion data are presented as mean ± SD of the adhesion frequency for individual cells (25 impingements each; n = 6–7 for control beads and 12–14 for CD80-Fc beads; p values for t tests between samples are shown).</p

Study sample diagram.

<p>Study sample diagram.</p

Sources of error per sample preparation method.

<p>(A) Intra-host single nucleotide variants (iSNVs) that were present in multiple samples at greater than 1% of population are shown in a heat map format to visualize patterned diversity acquired during sample preparation. The total number of samples containing iSNVs in greater than 1% of population are summarized in the column “#> 1% of Pop” in green. “Codon” column (second column from right) provides both the nucleotide and protein translation of the site. (B) The detected mean error rates of iSNVs greater than 0. 2% of population (error/site/copy) are stratified by presence in the plasmid (Origin), detection after transcription/reverse transcription (Transc) or preparation, and preparation/sequencer error (Prep). (C) Error profiles are expressed as the number of iSNVs per percent of population obtained from each of the 5 sample preparation methods.</p

Total error by sample preparation method.

<p>(A) The mean read depth per position from two DNA controls (PLASMID and P_AMP) and five RNA sample preparation methods (n = 2 of replicate experiments). (B) Errors calculated as error/site/copy (plasmid or transcript) are presented as the average of duplicate experiments as in A. Intra-host single nucleotide variants present in the original plasmid were removed from the calculation of the error (reference positions: 4,697 and 4,725). Student t-tests were performed to demonstrate differences between the mean error per site per copy in the control plasmid (* = <i>p</i><0.05) and each sample preparation method. (C) The error calculated in B is converted to fold change over the control (“PLASMID”). Error bars in all panels represent standard deviation of the mean.</p

Percentage of errors by type of acquired diversity determined during sample preparation.

<p>(A) Percentage of error attributed to synonymous vs. non-synonymous variants. (B) Percentage of errors attributed to transitions vs. transversions. (C) Percentage of errors attributed to insertions or deletions vs. Intra-host single nucleotide variants (iSNV).</p

Boletín de Segovia: Número 129 - 1841 octubre 28

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200