Supplemental Material - Engagement With Remote Delivery Channels in a Physical Activity Intervention for Senior Women in the US

Supplemental Material for Engagement With Remote Delivery Channels in a Physical Activity Intervention for Senior Women in the US by Andrea S. Mendoza-Vasconez, Abby C. King, Gabriel Chandler, Sally Mackey, Shawna Follis, and Marcia L. Stefanick in American Journal of Health Promotion.</p

BCellClassSwitching

Scripts associated with Horns et al. Elife 2016. Available in GitHub at https://github.com/felixhorns/BCellClassSwitchin

Defective T Memory Cell Differentiation after Varicella Zoster Vaccination in Older Individuals

<div><p>Vaccination with attenuated live varicella zoster virus (VZV) can prevent zoster reactivation, but protection is incomplete especially in an older population. To decipher the molecular mechanisms underlying variable vaccine responses, T- and B-cell responses to VZV vaccination were examined in individuals of different ages including identical twin pairs. Contrary to the induction of VZV-specific antibodies, antigen-specific T cell responses were significantly influenced by inherited factors. Diminished generation of long-lived memory T cells in older individuals was mainly caused by increased T cell loss after the peak response while the expansion of antigen-specific T cells was not affected by age. Gene expression in activated CD4 T cells at the time of the peak response identified gene modules related to cell cycle regulation and DNA repair that correlated with the contraction phase of the T cell response and consequently the generation of long-lived memory cells. These data identify cell cycle regulatory mechanisms as targets to reduce T cell attrition in a vaccine response and to improve the generation of antigen-specific T cell memory, in particular in an older population.</p></div

Mapping epitopes of U1-70K autoantibodies at single-amino acid resolution

<div><p></p><p>The mechanisms underlying development of ribonucleoprotein (RNP) autoantibodies are unclear. The U1-70K protein is the predominant target of RNP autoantibodies, and the RNA binding domain has been shown to be the immunodominant autoantigenic region of U1-70K, although the specific epitopes are not known. To precisely map U1-70K epitopes, we developed silicon-based peptide microarrays with >5700 features, corresponding to 843 unique peptides derived from the U1-70K protein. The microarrays feature overlapping peptides, with single-amino acid resolution in length and location, spanning amino acids 110–170 within the U1-70K RNA binding domain. We evaluated the serum IgG of a cohort of patients with systemic lupus erythematosus (SLE; <i>n</i> = 26) using the microarrays, and identified multiple reactive epitopes, including peptides 116–121 and 143–148. Indirect peptide ELISA analysis of the sera of patients with SLE (<i>n</i> = 88) revealed that ∼14% of patients had serum IgG reactivity to 116–121, while reactivity to 143–148 appeared to be limited to a single patient. SLE patients with serum reactivity to 116–121 had significantly lower SLE Disease Activity Index (SLEDAI) scores at the time of sampling, compared to non-reactive patients. Minimal reactivity to the peptides was observed in the sera of healthy controls (<i>n</i> = 92). Competitive ELISA showed antibodies to 116–121 bind a common epitope in U1-70K (68–72) and the matrix protein M1 of human influenza B viruses. Institutional Review Boards approved this study. Knowledge of the precise epitopes of U1-70K autoantibodies may provide insight into the mechanisms of development of anti-RNP, identify potential clinical biomarkers and inform ongoing clinical trails of peptide-based therapeutics.</p></div

Influence of pre-existing VZV immunity on vaccine responses.

<p>(A) VZV-specific T cell frequencies were determined by IFN-γ–specific ELISpot before (day 0) and at days 8±1, 14±1 and 28±3 after vaccination. **p<0.0001 by paired Wilcoxon-Mann-Whitney test. (B) Antibody indices determined by VZV-IgG-specific ELISA increased between day 0 and 28 (p<0.01). Data from individual vaccinees are joined with a line. (C and D) Fold change in VZV-specific antibody concentrations from day 0 to day 28 were negatively correlated with initial antibody concentrations (C, r² = 0.73, p <0.001), but not with initial VZV-specific T cell frequencies (D, p = 0.16). (E and F) Fold change in VZV-specific T cell frequencies from day 0 to day 28 showed no correlation with initial VZV-specific antibodies (E, p = 0.47) or initial T cell frequencies (F, p = 0.12). (G) Fold change in VZV-specific antibodies from day 0 to day 28 did not correlate with fold change in VZV-specific T cell frequencies from day 0 to day 28 (p = 0.38).</p

Contribution of initial expansion and subsequent contraction of VZV-specific T cells after vaccination to final T cell memory frequencies.

<p>VZV-specific T cell frequencies were measured by IFN-γ–specific ELISpot. Peak ELISpot counts were defined as the highest observed count at either day 8 (25/29 people) or day 14 (4/29 people) after vaccination. Absolute increase to the peak value (expansion) was calculated compared to day 0 counts. Absolute decline from peak (contraction) was calculated compared to day 28 counts. (A) Unsupervised hierarchical clustering analysis based on frequencies of VZV-specific T cells at all three time points. Vaccinees separated into three main clusters [C1 (black), C2 (red), C3 (blue)]. The color code for the different clusters is maintained in Fig 2B–2F. (B) Mean±SD of VZV-specific T cell frequencies at day 0, peak, and day 28 are shown for each cluster. (C-F) Boxplots showing overall increases in VZV-specific T cell frequencies from day 0 to day 28 (C), expansion in VZV-specific T cell frequencies from day 0 to peak value (D), contraction in VZV-specific T cell frequencies from peak to day 28 (E), and age range of individuals in each cluster (F). p-values calculated by one-tailed Wilcoxon-Mann-Whitney test are shown. (G) Vaccinees were grouped according to their age < 59 (orange) or >59 (green) years and T cell frequency trajectories after vaccinations are shown as described in Fig 2B. (H) When corrected for age, VZV-specific T cell expansion showed a weak correlation with overall increase in VZV-specific T cell frequency from day 0 to day 28 that did not reach significance (r = 0.31, p = 0.10). (I) Contraction after peak responses corrected for age inversely correlated with overall increase in VZV-specific T cell frequency from day 0 to day 28 (r = -0.53, p = 0.003).</p

Correlation of whole blood-derived gene signatures with VZV-specific T cell responses.

<p>(A) Deconvolution of whole blood gene expression for leukocyte subsets was performed. Volcano plots show fold change of gene expression in monocytes (left), lymphocytes (middle) and neutrophils (right) between day 0 and day 1. The genes with significant changes in expression (fold change>1.5, p<0.05) after vaccination are colored in red. (B) Fold changes in monocyte-derived genes (day 0 to day 1, based on deconvolution analysis) were correlated with log-transformed changes in frequencies of antigen-specific T cells after VZV vaccination. Results are shown as volcano plots of correlation coefficients for VZV-specific T cell expansion (VZV-specific T cell frequencies day 0 to peak, left panel), contraction (peak to day 28, middle panel) and overall responses (day 28 to day 0, right panel). The genes with significant correlations (p<0.05) are colored in red. (C) The Venn diagram shows the overlap in genes that significantly correlate with expansion, contraction or global responses.</p

Lasso cytokine predictors of antigen-specific T cell expansion and contraction after VZV vaccination.

<p>(A) Principal component analysis of serum concentrations of 51 cytokines on day 0 and day 1 was performed. The box plots show the log transformed fold changes (day 1 to day 0) of PC1 (explaining 60% of variation, left panel) and PC2 (9%, right panel) among individuals. PC2 significantly changes between before and day 1 after vaccination (p = 0.017). (B-E) Panels B and D show the estimated mean square error (y-axis) from a sequence of lasso models in predicting VZV-specific T cell expansion (B) and contraction (D) using the baseline frequencies of VZV-specific T cells and serum cytokine changes between day 0 and day 1 after vaccination. The x-axis represents the log-transformed penalty parameter controlling the model complexity determined by the number of predictors in the model shown on top. Panels C and E plot the predicted vs. the true frequencies after leave-one-out cross-validation for the lasso procedures for VZV-specific T cell expansion (C) and contraction (E). The 45° line is shown for orientation.</p

T cell responses to VZV vaccination are more similar in identical twins than in non-twins.

<p>Pairwise comparisons of fold changes in VZV-specific T cell frequencies (A, C, D) and antibodies (B) were performed for all individuals. Histograms show the frequency distributions of the differences in fold changes for each pair of individuals. Red lines show the position of identical twin comparisons. (A) T cell responses (day 28 to day 0) were more similar between identical twins than between unrelated individuals (p = 0.008). (B) Antibody responses were no more similar between twins than between unrelated individuals (p = 0.44). (C) Effector cell expansion (day 0 to peak) in twins was more similar compared with non-twins with two notable outliers, therefore not reaching significance (p = 0.28). (D) Contraction (peak to day 28) was slightly more similar between twins than between non-twins without reaching significance (p = 0.16).</p

Demographics of study participants.

<p>Demographics of study participants.</p