Additional file 1: of Risk factors for COPD exacerbations in inhaled medication users: the COPDGene study biannual longitudinal follow-up prospective cohort

Supplementary Data and Methods. (DOCX 38 kb

HLA-C fractional synthesis in unstimulated MoDCs.

<p>Analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.g006" target="_blank">Fig 6</a>.</p

HLA-B fractional synthesis in unstimulated MoDCs.

<p>Analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.g006" target="_blank">Fig 6</a>, except that separate curve fits are shown for HD2 (p = 0.04, F test). The significance of this result is doubtful, as explained in the text. B*27+ donors are identified; note that B27 allele-specific peptides proved suitable for analysis in HD6 and 7, but not in HD4 and 5.</p

MHCI turnover in KG-1 cells.

<p>Proliferating KG-1 cells were labeled with ≈ 5% ²H₂O in media and MHCI molecules immunoprecipitated with W6/32. ²H incorporation into selected peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.s003" target="_blank">S1 Table</a>; identified here by the four N-terminal amino acids and assigned to isotypes and alleles as shown) was quantified by LC-MS. One of two independent experiments is shown here; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.s003" target="_blank">S1 Table</a> summarizes results for both. (A-C) Fractional synthesis was calculated for different peptides derived from HLA-A (A), HLA-B (B), and HLA-C molecules (C) (mean ± SD of the informative mass isotopomers) and plotted against time. In (B) and (C), allele- and isotype-specific peptides exhibited no significant differences in fractional protein synthesis (p = 0.24 and p = 0.55, respectively, by F test). Single-exponential curve fits (with 95% confidence intervals) are based on a pooled analysis of all peptides from each isotype. (D) Exponential growth of KG-1 cells during ²H₂O labeling. The corresponding time course of the fraction of new cells is shown in panels (A-C) for comparison with protein synthesis. (E) Fractional synthesis rates (per hour, mean ± SEM) of MHCI isotypes (from (A-C)), compared with cell growth (from (D)). The differences between HLA-C and the other isotypes, and those between the MHCI fractional synthesis rates and cell growth, were significant (each p < 0.0001, F test). (F) Turnover half-lives of different MHCI isotypes, calculated from the excess of mean fractional protein synthesis rates over the cell growth rate.</p

MHCI turnover in LCL721 cells.

<p>LCL721 cells were labeled with ≈ 5% ²H₂O in media and folded MHCI molecules immunoprecipitated with W6/32. ²H incorporation into tryptic peptides was quantified by LC-MS. (A-C) Fractional synthesis was calculated for different peptides derived from HLA-A (A), HLA-B (B), and HLA-C molecules (C) (mean ± SD of different mass isotopomers) and plotted against labeling time. Full sequences and analytical metrics for all informative peptides (identified by four amino acids in single-letter code or by charge) are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.s004" target="_blank">S2 Table</a>. In (A) and (B), allele- and isotype-specific peptides exhibited no significant differences in fractional protein synthesis (p = 0.32 and p = 0.29, respectively, by F test); in (C), only C1-specific peptides were identified. Single-exponential curve fits (with 95% confidence intervals) are based on a pooled analysis of all peptides from each isotype. (D) Exponential growth of LCL721 cells during ²H₂O labeling. The corresponding time course of the fraction of new cells is shown in panels (A-C), for comparison with protein synthesis. Panels (A-D) were from the same experiment. (E) Fractional synthesis rates (per hour, mean ± SEM) of MHCI isotypes (from (A-C)), compared to fractional cell growth rates (from (D)). Two independent experiments are shown. Fractional synthesis rates for individual peptides are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161011#pone.0161011.s004" target="_blank">S2 Table</a>. (F) Turnover rates of different MHCI isotypes, calculated from the excess of mean fractional protein synthesis rates over the cell growth rate.</p

Effect of ²H₂O labeling on peptide mass isotopomer distributions.

<p>(A) Mass isotopomer distributions of a MHCI-derived, B isotype-specific tryptic peptide from KG-1 cells after labeling with ²H₂O for various times. Lines connect data at each time point. Within error, each mass isotopomer changed from its initial (unlabeled) to final plateau (fully-labeled) value at the same rate, which is identical to the rate of protein fractional synthesis. (B) For the same peptide, MIDA models for the unlabeled and fully-labeled mass isotopomer distributions (dashed and solid lines, respectively) were compared with experimental data (symbols). RMSD values were 0.20% and 0.25%, respectively, for unlabeled and fully-labeled samples).</p

Summary of fractional synthesis rates of MHCI isotypes in MoDCs.

<p>(A) Scheme illustrating MoDCs differentiation, followed by mock or LPS (100 ng/ml) stimulation, with subsequent ²H₂O labeling for 72 hours, beginning 24 hours after LPS treatment. (B) Comparison between turnover rates of MHCI isotypes in individual unstimulated MoDC cultures from 8 HDs and one B27-negative AS patient. In this setting, fractional synthesis was taken to equal turnover (see text). (C) Effect of LPS on fractional synthesis rates of MHCI isotypes in two HDs.</p

Measuring MHCI protein turnover by ²H₂O labeling.

<p>(A) SINEW work flow. See text for details. (B) New protein synthesis may support cell growth, increase net protein levels per cell, or replace protein lost to turnover. These processes contribute additively to protein synthesis. (C) Assignment of peptides to particular MHCI alleles or isotypes. First, LC-MS/MS data (<i>Step 4</i> in panel A) are screened against sequence databases to identify tryptic fragments derived from <i>any</i> MHCI molecules (<i>top</i>, color-coded boxes). MHCI alleles present in each donor are identified by HLA genotyping, and their predicted amino acid sequences are subjected to tryptic digestion <i>in silico</i>. These virtual digests are compared with each other to identify a subset of peptides that are specific to particular alleles or isotypes (symbolised by boxes with text labels).</p

Increased B-cell activation in mesenteric lymph nodes of NOD^high mice.

<p>(<b>A</b>) Total cell counts in the indicated lymphoid organs of NOD^low (closed circles) vs. NOD^high (open circles) mice: MLN = mesenteric lymph nodes, PLN = pancreatic lymph nodes, ILN = inguinal lymph nodes. Analysis by 2-way ANOVA with p values given for significant colony differences. (<b>B</b>) Representative flow cytometry dot plots of CD69^high B cells isolated from mesenteric lymph nodes of NOD^low (left) and NOD^high (right) females at six weeks of age. (<b>C</b>) Frequencies of CD69^high cells in gated B220⁺ B cells from mesenteric lymph nodes (MLN) or spleens (SP) of NOD^low (closed circles) vs. NOD^high (open circles) female mice. Individual mice, means and significant p values (p < 0.05, by 2-way ANOVA) are shown.</p

T1D is induced in NOD^low mice by anti-PD-L1 antibody, but not by cyclophosphamide.

<p>(<b>A</b>) Normoglycaemic (pre-diabetic) NOD^low (black circles) and NOD^high (white circles) female mice were treated at 16–18 weeks of age with cyclophosphamide, and T1D development was monitored daily up to 25 days post intra-peritoneal injection. (<b>B</b>) Representative FACS plots for CD4 (x-axis) vs. FoxP3 (y-axis) on gated CD4⁺ T cells from NOD^low (left) and NOD^high (right) spleens. (<b>C</b>) Percentages (left) and absolute numbers (right) of FoxP3⁺ CD4 T cells from pancreatic lymph nodes of NOD^low (full circles) and NOD^high (white circles) mice. Statistical analysis was performed using Student’s t test. (<b>D</b>) Normoglycaemic (pre-diabetic) NOD^low females were treated with anti- (α-) PD-L1 (clone MIH5, grey circles) or PBS (black circles) at 12–14 weeks of age, and T1D development was monitored daily up to 25 days post intra-peritoneal injection. In (A) and (D), diabetes-free survival was compared between groups by Kaplan-Meier analysis and log rank test.</p