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Blood, Vol. 113, Issue 25, 6465-6476, June 18, 2009
Impact of donor CMV status on viral infection and reconstitution of multifunction CMV-specific T cells in CMV-positive transplant recipients
Blood Zhou et al.
113: 6465
Supplemental materials for: Zhou et al
Files in this Data Supplement:
- Figure S1. Time to first GCV use (JPG, 246 KB)
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Kaplan-Meier estimates show the probability of avoiding the need for antiviral therapy as a function of time, comparing D+/R+ with D−/R+ recipients. The logrank test result (p=0.05) is not robust to adjustment for baseline covariates (p=0.18), but the comparison is significant when accounting for repeated events (see Fig. 1B).
- Figure S2. Gating scheme for measuring cytokine and CD107 expression on PBMC (JPG, 429 KB)
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Shown are gating schemes for identification of multi-functional CD8+ T cells and representative data at d180 post-HCT. (A) Representative flow results from D+/R+ patients after ex vivo stimulation with pp65 peptide library. PBMC were gated for lymphocyte population based on forward and side scatter followed by CD3+ CD8+ selection. (B) CD8+ T cells from D+/R+ patients were then gated for IFN-γ and MIP-1β combinations. IFN-γ+ MIP-1β+ cells (upper right quadrant) and IFN-γ+ MIP-1β− cells (lower right quadrant) were gated separately and analyzed for expression of TNF-α and CD107. Thus 8 combinations of single, double, triple and quadruple functional subsets were identified, and the percentage of each subset within the total population of functional CD8+ T cells was calculated. For example: the percentage of IFN-γ+ MIP-1β+ TNF-α+ CD107+ CD8+ T cells = 14.15 × 3.45/ (3.45+2.04) = 13.6. Flow results from D−/R+ group patients are shown in (C). In both groups, mock stimulations were used as negative controls.
- Figure S3. Single Cytokine (IFN-γ) measurements of CD4+ T cells in a subset of Group 2 patients (JPG, 335 KB)
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In a preliminary study, patients randomly selected from among Group 2 patients (Table 1) evaluated for IFN-γ–CD8+ were also evaluated for levels of IFN-γ expressed in CD4+ T cells using a similar approach described in the legend of Fig. 2. The only difference was substitution of the CD4 cell-surface mAb instead of CD8. Sample limitations precluded an exhaustive analysis of all patients at all timepoints, therefore an unequal number of samples were evaluated at d90, 180, and 360 solely based on sample availability. This limitation prevented formal statistical analysis, including univariate group comparisons at each timepoint, or the formal multivariate analysis applied to measurements of CD8+ T-cell function shown in Table 3. The number of patients with either a D+ or D− donor at each timepoint is shown in parentheses. There was a minimum of 2-fold difference in IFN-γ levels in patients with D+ donors at each timepoint (see averages at top of figure), but no statistical significance was calculated because of the unequal sample sizes.
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