MOESM2 of Clinical implications of CD4+ T cell subsets in adult atopic asthma patients

Additional file 2: Figure S1. Correlation analysis of frequency of CD4+ and CD8+ memory T cells with age of recruited subjects

DataSheet_1_Disruption of endosomal trafficking with EGA alters TLR9 cytokine response in human plasmacytoid dendritic cells.pdf

Plasmacytoid dendritic cells (pDCs) exhibit bifurcated cytokine responses to TLR9 agonists, an IRF7-mediated type 1 IFN response or a pro-inflammatory cytokine response via the activation of NF-κB. This bifurcated response has been hypothesized to result from either distinct signaling endosomes or endo-lysosomal trafficking delay of TLR9 agonists allowing for autocrine signaling to affect outcomes. Utilizing the late endosome trafficking inhibitor, EGA, we assessed the bifurcated cytokine responses of pDCs to TLR9 stimulation. EGA treatment of pDCs diminished both IFNα and pro-inflammatory cytokine expression induced by CpG DNAs (D- and K-type), CpG-DNAs complexed with DOTAP, and genomic DNAs complexed with LL37. Mechanistically, EGA suppressed phosphorylation of IKKα/β, STAT1, Akt, and p38, and decreased colocalization of CpG oligodeoxynucleotides with LAMP+ endo-lysosomes. EGA also diminished type 1 IFN expression by pDCs from systemic lupus erythematosus patients. Therefore, our findings help understand mechanisms for the bifurcated cytokine responses by pDCs and support future examination of the potential benefit of EGA in treating type 1 IFN-associated inflammatory diseases in the future.</p

The B cell response following infection with different viruses that replicate in the lung.

<p>(A–F) Virus-specific ASC frequencies. DR1 (A–C) and B10 (D–F) mice were infected intranasally with the influenza viruses PR8 (A and D) and X31 (B and E), and with the non-influenza virus MHV68 (C and F). Virus-specific ASC frequencies in the MedLN were determined by ELISPOT assay at intervals after infection. (G) PR8 replication in the lung. Titers are represented as log₁₀ TCID₅₀/0.2 ml of lung homogenate. (H, I) Flow cytometric analysis of ASCs and germinal center B cells in the MedLN on day 8 after PR8 infection. ASC frequencies (H) represent the proportion of B220^int CD138⁺ cells after gating on live CD4⁻ CD8⁻ CD19⁺ cells. Germinal center B cell frequencies (I) represent the proportion of PNA⁺ Fas⁺ cells among live CD4⁻ CD8⁻ B220⁺ cells. (J) Serum levels of virus-specific IgG in DR1 and B10 mice on day 8 after PR8 infection. Titers determined by ELISA are shown as the reciprocal of the highest serum dilution scored as positive relative to naïve control serum. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone-0034377-g006" target="_blank">Fig. 6</a> data sets depict the mean+SE for 3–10 individual mice per group. * P<0.05, *** P<0.001.</p

The CD4 T cell response to NC infection.

<p>(A–D) Cytokine production by peptide-specific CD4 T cells. DR1 (A and B) and B10 (C and D) mice were infected intranasally with 40,000 EID₅₀ NC. Enriched CD4 T cells from the MedLN and spleen were analyzed on day 10 after infection. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and individual 17-mer peptides. Peptide designations (x-axis) include the viral proteins of origin (HA, NA, NP, M1, and NS1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean+SEM for 2–6 independent experiments for each peptide. Cells from at least three mice were pooled for each experiment. (E, F) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in A–D and represent 4 (MedLN) or 6 (spleen) independent experiments evaluating 3–12 (MedLN) or 12–20 (spleen) individual peptides. The mean+SEM is shown.</p

Cytokine and chemokine production in the lung after influenza infection.

<p>DR1 and B10 mice were infected intranasally with NC or PR8 or were mock-infected with PBS. Mice were sampled 60 h after inoculation. Cytokine and chemokine concentrations in clarified lung homogenates were determined by Multiplex assay or by sandwich ELISA (IFN-α only). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone-0034377-g009" target="_blank">Figure 9</a> shows the results for a selection of the 30 cytokine and chemokine determinations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> for the remaining cytokines and chemokines are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#pone.0034377.s004" target="_blank">figure S4</a>. The mean+SE is shown for 5 individual mice per group. * P<0.05, ** P<0.01, *** P<0.001.</p

The virus-specific B cell and CD4 T cell response to intramuscular immunization.

<p>(A–D) Virus-specific ASC frequencies. DR1 (A and B) and B10 (C and D) mice were immunized intramuscularly with inactivated NC. Virus-specific ASC frequencies in the iliac lymph nodes and spleen were determined by ELISpot assay at the indicated times after immunization. The mean+SE is shown for 3–4 individual mice per group. (E, F) Cytokine production by peptide-specific CD4 T cells. Enriched CD4 T cells from the spleen were analyzed on day 8 after immunization. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and sets of 1–3 17-mer peptides from different viral proteins (HA, NA, NP, and M1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean ± range for 2 independent experiments (the M1 protein was represented in only one experiment with B10 mice). Cells from at least three mice were pooled for each experiment. (G, H) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in E and F. The mean ± range is shown for the two independent experiments.</p

Serum levels of virus-specific Ab in DR1 and B10 mice after NC infection.

<p>NC-specific IgM (A), IgG1 (B), IgG2b (C), IgG2c (D), IgG3 (E), IgA (F), IgG (G), and Ig (H) titers were determined by ELISA on plates coated with disrupted viral particles. Titers are shown as the reciprocal of the highest serum dilution scored as positive relative to naïve control serum. The mean+SE is shown for 4–8 individual mice per group. * P<0.05, ** P<0.01.</p

The CD4 T cell response to PR8 infection.

<p>(A, B) Cytokine production by peptide-specific CD4 T cells. DR1 and B10 mice were infected intranasally with PR8. Enriched CD4 T cells from the MedLN were analyzed on day 10 after infection. Frequencies of CD4 T cells secreting IL-2, IFN-γ, or IL-4 were determined by ELISpot assay after in vitro stimulation with antigen-presenting cells and individual 17-mer peptides. Peptide designations (x-axis) include the viral proteins of origin (NP, M1, and NS1). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are normalized to spot counts per 10⁶ CD4 T cells and are shown as the mean ± range for 2 independent experiments. Cells from at least three mice were pooled for each experiment. (C, D) Proportions of peptide-specific CD4 T cells secreting IL-2, IFN-γ, or IL-4. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034377#s3" target="_blank">Results</a> are compiled from the data shown in A and B. The mean ± range is shown for the two independent experiments.</p

DataSheet_1_Endosomal trafficking inhibitor EGA can control TLR7-mediated IFNα expression by human plasmacytoid dendritic cells.pdf

Plasmacytoid dendritic cells (pDC) are the major producer of type 1 IFN in response to TLR7 agonists. Aberrant TLR7 activation and type 1 IFN expression by pDCs are linked to the pathogenesis of certain types of autoimmune diseases, including systemic lupus erythematosus (SLE). This study investigated the underlying mechanisms for TLR7-mediated cytokine expression by pDCs using a late endosome trafficking inhibitor, EGA (4-bromobenzaldehyde N-(2,6-dimethylphenyl) semicarbazone). We found that EGA treatment decreased IFNα expression by pDCs stimulated with imiquimod (R837), single-stranded RNA40, and influenza virus. EGA also decreased TNFα expression and secretion by R837-stimulated pDCs. Mechanistically, EGA treatment decreased phosphorylation of IKKα/β, STAT1, and p38, and prolonged degradation of IκBα. Furthermore, EGA treatment decreased the colocalization of 3F, a substituted adenine TLR7 agonist, with LAMP1+ compartments in pDCs. EGA was also capable of diminishing IFNα expression by SLE pDCs treated with R837 or live PR8/A/34 influenza viruses. Therefore, we concluded that trafficking of TLR7 agonists to LAMP1+ compartments is important for IFNα expression by pDCs. Data from this study support additional examinations of the potential benefits of EGA in treating type 1 IFN-associated inflammatory diseases in the future.</p