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. 2022 Apr 13;13:805026. doi: 10.3389/fimmu.2022.805026

Figure 2.

Figure 2

Tetanus toxoid-reactive CD154+ T helper cells increased after vaccination. Six horses were booster vaccinated against tetanus toxoid on day 0 and antigen-reactive lymphocytes in peripheral blood were analyzed before (0) and on days 7, 14 and 28 after vaccination. PBMC were re-stimulated with tetanus toxoid (TT) as an antigen in vitro, compared to medium incubation for 5h. (A) Pseudocolor plots of a representative sample (day 14) illustrate the gating strategy and data reduction. Doublets, and dead cells (live-dead (LD) staining) were excluded, and lymphocytes were gated by forward scatter (FSC) and side scatter (SSC) characteristics resulting in a population of (singlet live) lymphocytes. Percentages of cell populations (e. g. CD4+CD154+) were quantified in quadrant gates (% of lymphocytes). To analyze stimulation effects, baseline CD154 expression in medium (blue) was subtracted from that in stimulated (TT, red) lymphocytes resulting in (medium-corrected) net % of lymphocytes (green). (B–E) Net % of lymphocytes of (B) CD154+, (C) CD4+CD154+, (D) CD4-CD154+, and (E) CD8+CD154+ are plotted over time. The connecting line depicts medians of n=6 (day 28 n=4). Significant differences (p<0.05) after vaccination compared to pre-vaccination frequencies are indicated as * for CD4+CD154+ on day 14.