has received compensation as a member of the Scientific Advisory Board on dengue vaccines for Sanofi Pasteur. significant. RESULTS CD4 and CD8 T-Cell Expansion During Acute Dengue Illness To investigate T-cell activation in vivo, we analyzed 116 PBMC samples obtained from 27 Thai children during and after acute DENV infection using multi-parametric flow cytometry. Nine and 18 patients were diagnosed with primary (1) and secondary (2) DENV infections, respectively. Nineteen patients had DF and 8 patients had DHF. A summary of the patient cohort information is found in Supplementary Table S1. The PBMC samples were collected at febrile (fever days ?5 to ?1), critical (fever days 0 to +1), early convalescence (fever days +3 to +8), and healthy (6 months to 2 years postenrollment) time points. Figure 1A shows our gating strategy to identify CD4 and CD8 T cells. We found an increase in CD8 frequencies coincident with a decrease in CD4 frequencies during acute infection (Figure 1B). Although the frequencies of CD4 T cells decreased, the average numbers of CD4 and CD8 T cells both increased during acute DENV infection (Figure 1C). Open in a separate window Figure 1. Expansion of CD4 and CD8 T Citicoline sodium cells during the course of dengue infection. The gating strategy for the flow cytometry analysis of CD4 and CD8 T-cell Citicoline sodium subsets is shown (A). The frequency of CD8 (filled circles) and CD4 (open circles) T cells is shown during febrile, critical, early convalescence (E. C.) and healthy time points. Generalized estimating equation models were also used to determine the statistical trend for an increase in CD8 and decrease in CD4 frequencies from fever day ?5 to E. C. (B). The number of CD8 (filled circles) and CD4 (open circles) T cells is shown during febrile, critical, E. C. (C). Horizontal lines represents the median for all data points, and bars indicate the interquartile range. *, .05; ****, .0001. CD4 and CD8 T Cells Are Highly Activated During Acute Dengue Illness To study the kinetics of T-cell activation, we used antibodies against CD38 and PD-1 because these markers are elevated on CD8 T cells in PBMCs from patients DCHS1 undergoing acute DENV infection [15, 18, 19]. We found significant PD-1 and CD38 coexpression on both CD8 and CD4 T cells during the febrile, critical, and early convalescence phases of infection when compared with samples obtained from the same individuals 6 months to 2 years later (Figure 2). The mean frequencies of activated (PD-1+ and CD38+) CD8 and CD4 T cells (Figure 2B) were highest during the critical phase of illness (44% and 18%, respectively). We wanted to determine whether there are significant differences in the number of activated CD8 and CD4 T cells in patients with primary versus secondary DENV infections and DF versus DHF. During the critical phase of illness (fever day 0 to +1), the mean (2:5.44, 1:4.82, 2:4.67, and 1:4.16 log10 cells/mL) and median (2:5.46, 1:4.77, 2:4.80, and 1:4.21 log10 cells/mL) number of activated (PD-1+ and CD38+) CD8 and CD4 T cells, respectively, were significantly higher in patients with secondary versus primary DENV infections (Figure 2C). When comparing patients with DF and DHF, we found the mean (DHF:5.53, DF:5.18, DHF:4.73, DF:4.43 log10 cells/mL) and median (DHF:5.56, DF:5.33, DHF:4.89, and DF:4.53 log10 cells/mL) number of activated CD8 T cells to be Citicoline sodium significantly higher, but this difference did not reach statistical significance in CD4 T cells during the critical phase of illness (Figure 2D). Open in a separate window Figure 2. Robust activation of CD4 and CD8 T cells during acute.