Fig 2: Application of the T-cell panel tube in healthy controls and PID patients with T-cell defects. Analysis of circulating T-cell subsets (FS/SSloCD3+) identified within 1 × 106 blood leukocytes using the markers from the T-cell panel tube: naïve (CD45RO− CCR7+ CD27+ CD28+), Central Memory (CM) (CD45RO+ CCR7+ CD27+ CD28+), Transitional Memory (TM) (CD45RO+ CCR7− CD27+ CD28+/−), Effector Memory (EM) (CD45RO+ CCR7− CD27− CD28+), Terminal Effector (TE) (CD45RO− CCR7− CD27+ CD28+/−), and Terminally Differentiated (TD) (CD45RO− CCR7− CD27− CD28+/−) CD4+ and CD8+ T-cells. (A) The identification of biologically relevant subsets of T-cells in six healthy controls using the minimum number of bivariate plots required for a 6 marker-combination (three bivariate plots/cell population). (B) Comparable data via PCA1 vs. PCA2 representation of a 6-dimensional space in blood of six healthy controls (left) as compared to blood samples of three severe combined immunodeficiency (SCID) patients, diagnosed with RAG2, PNP, and DOCK8 defects, all stained with the RTE/SICD tube under comparable conditions. The SCID patients show “leakiness” with absence of naïve T-cells (<1 cell/μL blood) and variable “right shift” to mature CM/TM, EM, and TD T-cells in the CD4 lineage (all 3 SCID patients) and CD8 lineage (PNP and DOCK8 defects).

Fig 3: DNA/LION vaccination affects CD8 cell subpopulations(A) Pie charts depict the mean frequency in blood and LN of manually gated TEM (CD28+CD95+), central memory (TCM; CD28−CD95+), and naive (CD28+CD95−) CD8+T cell subsets and the circulating CXCR5+PD-1int/high CD8 cTfc1 and the two LN-associated CXCR5+PD-1int CD8 Tfc2 and CXCR5+ PD-1high CD8 Tfc3 subsets.(B) Heatmap shows manually gated Log2FC estimates from GEE of Ki67+ and Ki67+GzmB+ TCM and TEM in blood and LN. p values from GEE are summarized in the left columns with significant increase (red) and decrease (blue) with >2x and <2x upregulation shown in respective shades.(C) Heatmap shows manually gated Log2FC of CD8 cTfc1 in blood and CD8 Tfc1 in LN. Statistical analysis as in (B).(D and E) Bubble plots summarizing Spearman correlation (p = 0.05)of changes in cell populations (Log2FC D3/4vsD1; D8vsD1, D22vsD1; indicated by different bubble sizes) and changes of plasma cytokines/chemokines (log2FC, D2 vs. D1) for (D) TCM and TEM and (E) CD8 cTfc1 in blood and CD8 Tfc1 in LN.

Fig 4: DNA/LION vaccine induces durable T cell responses in rhesus macaques(A and B) Spike-specific T cell responses were measured in PBMCs over time by flow cytometry in (A) study 1, as IFN-γ + T cells, and (B) study 2, as cytokine+ (IFN-γ, TNF-α single- and/or double-positive) T cells. Scatterplots with median line and minimum and maximum values are shown. The pie chart depicts the frequency of spike-specific IFN-γ, TNF-α single- and/or double-positive T cells.(C) Comparison of CD4+ and CD8+ IFN-γ+ spike-specific T cell responses induced by DNA/LION (study 1, group 1) and by DNA(IM) prime followed by DNA/LION boost (study 1, group 2) and a historical study32 examining spike DNA vaccination by EP alone (n = 4) or co-immunized with adjuvanted spike protein (n = 4).(D) Frequency of spike-specific cytokine+ T cells CD4+ and CD8+ T cells at the day of V4 and 2 and 3 weeks later (study 1). Bars indicate mean values. p values from generalized estimating equation (GEE) and are defined as ≤0.05, ∗; ≤0.01, ∗∗; ≤0.001, ∗∗∗; ≤0.0001,∗∗∗∗.(E) Frequency of cytokine+ spike-specific CD8+ TCM (CD28+ CD95+) and TEM (CD28− CD95+ CCR7−) after V4. Scatterplots with mean values are shown.(F) Proportion of cytokine+ CD8+ T cells expressing EOMES and/or T-bet (mean of 8 animals).(G) Manually gated spike-specific cytokine+ CD107a+ CD8+ TCM and TEM cells (V4 [blue], V4w2 [red], V4w3 [green]) were overlaid in histograms depicting expression of EOMES, T-bet, GzmB, and Ki67 (mean fluorescent intensity [MFI] as mean of 8 animals at each time point). Gray dotted line marks the border between the negative and positive population.