Specificity and Dynamics of Effector and Memory CD8 T Cell Responses in Human Tick-Borne Encephalitis Virus Infection

<div><p>Tick-borne encephalitis virus (TBEV) is transferred to humans by ticks. The virus causes tick-borne encephalitis (TBE) with symptoms such as meningitis and meningoencephalitis. About one third of the patients suffer from long-lasting sequelae after clearance of the infection. Studies of the immune response during TBEV-infection are essential to the understanding of host responses to TBEV-infection and for the development of therapeutics. Here, we studied in detail the primary CD8 T cell response to TBEV in patients with acute TBE. Peripheral blood CD8 T cells mounted a considerable response to TBEV-infection as assessed by Ki67 and CD38 co-expression. These activated cells showed a CD45RA-CCR7-CD127- phenotype at day 7 after hospitalization, phenotypically defining them as effector cells. An immunodominant HLA-A2-restricted TBEV epitope was identified and utilized to study the characteristics and temporal dynamics of the antigen-specific response. The functional profile of TBEV-specific CD8 T cells was dominated by variants of mono-functional cells as the effector response matured. Antigen-specific CD8 T cells predominantly displayed a distinct Eomes+Ki67+T-bet+ effector phenotype at the peak of the response, which transitioned to an Eomes-Ki67-T-bet+ phenotype as the infection resolved and memory was established. These transcription factors thus characterize and discriminate stages of the antigen-specific T cell response during acute TBEV-infection. Altogether, CD8 T cells responded strongly to acute TBEV infection and passed through an effector phase, prior to gradual differentiation into memory cells with distinct transcription factor expression-patterns throughout the different phases.</p></div

Transcription factor profile of TBEV-specific cells.

<p>(A) Plots are gated on total CD8 T cells (black background) or TBEV-specific (NS3 ILL) CD8 T cells (green dots). (B) Bar plots show the median and 10–90th percentiles of each marker in ILL⁺ specific cells from five donors. (C) Bar plots show the median and 10–90th percentiles of each marker in total CD8 T cells from five donors (D) Bar chart represents subset distribution of T-bet, Eomes and Ki67 in TBEV-ILL-specific cells. (E) Bar chart represents subset distribution of T-bet, Eomes and Ki67 in total CD8 T cells. (F) Median and 10–90th percentiles of Eomes⁺Ki67⁺T-bet⁺ total CD8 T cell subset at day 7, 21 or 90 after hospitalization in infected subjects (n = 10) together with healthy controls (n = 16). (G) Median and 10–90th percentiles of granzyme B and perforin in Eomes⁺ and Eomes⁻ total CD8 T cell subsets at day 7 after hospitalization. Statistical analysis was performed by using non-parametric repeated measures ANOVA test or Mann-Whitney test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Functional profile of TBEV-specific effector and memory CD8 T cell responses.

<p>(A) PBMCs from infected subjects (n = 5) and healthy controls (n = 5) were stimulated for 6 hours with a pre-selected TBEV peptide pool in the presence of brefeldin A and monensin. Intracellular expression of MIP-1β, IFN-γ, and TNF, as well as the cell surface expression of CD107a, were assessed by flow cytometry and are shown as respresentative flow plots at day 21 after hospitalization. (B) Bar plots show the 10–90th percentiles of CD107a, MIP-1β, TNF and IFN-γ production in response to a pre-selected peptide pool at day 0, 7, 21 and 90 after hospitalization (n = 5). (C) Pie charts indicate the mean composition of the total response in CD8 T cells with regards to their capacity to express one, two, three or four functions at days 7, 21 and 90 after hospitalization. (D) Dominant polyfunctional profiles at day 7, 21 and 90 after hospitalization. The percentage of parent populations is indicated for each dominant population. (E) Bar chart represents the subset distribution of CD27, CD45RA and CD57 in cells responding with CD107a, MIP-1β, IFN-γ, or TNF after stimulus with a pre-selected peptide pool day 7, 21 and 90 after hospitalization. (F) Heat map represents subset distribution of Eomes and T-bet in cells responding with CD107a, MIP-1β, IFN-γ, or TNF after stimulus with the pre-selected peptide pool at day 0, 7, 21 and 90 after hospitalization. Statistical analysis was performed by using the non-parametric repeated measures ANOVA test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Transcriptional profile of activated CD8 T cells.

<p>(A) Flow plots show Helios, T-bet and Eomes stainings in CD8 T cells from one representative donor. (B) Bar plots show the 10–90th percentiles of T-bet, Eomes and Helios expression in the CD38 and Ki67 co-expressing CD8 T cell subset at day 7 after hospitalization (n = 10), and at day 7 after hospitalization in non-activated cells or in non-activated healthy controls (n = 16). (C) Heat map represents subset distribution of Eomes and T-bet within the CD38 and Ki67 co-expressing CD8 T cells (n = 10). Statistical analysis was performed using the Mann-Whitney test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Activation of T cells in the acute phase of TBE infection.

<p>(A) CD38 and Ki67 co-expressing cells in the total CD8 T cell population over time in one representative patient. (B) Median and 10–90th percentiles of CD38 and Ki67 co-expression in CD8 T cell subsets at day 0, 7, 21 and 90 after hospitalization in infected subjects (n = 20) and in healthy controls (n = 20). (C) Ki67 expression vs CMV-pp65 HLA MHC class I tetramer staining over time in one donor. Percent Ki67⁺ CMV pp65⁺ cells are indicated in the plot. (D) Kinetics of Ki67 expression in CMV⁺ (red line) and CMV⁻ (black line) CD8 T cells in four donors over time. (E) Stainings of perforin, CD45RA, PD-1, Bcl-2, CD127, granzyme B, CD27 and HLA-DR at day 7 after hospitalization. Gated on total CD8 T cells. (F) Bar plots show the 10–90th percentiles of HLA-DR, Bcl-2, PD-1, granzyme B, perforin and CD127 expression together with CD27 in terms of mean fluorescence intensity in CD38 and Ki67 co-expressing CD8 T cell subset at day 7 after hospitalization, non-activated Ki67⁻CD38⁻ (N-A) cells at day 7 after hospitalization or in non-activated healthy controls (N-A HC). (G) Bar chart represents the subset distribution of CCR7, CD45RA and CD127 (IL7Rα) in CD38 and Ki67 co-expressing cells at day 7 after hospitalization. (H) CD38 and Ki67-coexpressing cells in CD4 cell population over time in one infected patient. (I) Median and 10–90^th percentiles of CD38 and Ki67 co-expression in CD4 T cell subset at the day of hospitalization (day 0) and at day 7, 21 and 90 after hospitalization in infected subjects (n = 20) together with healthy controls (n = 20). Statistical analysis was performed using non-parametric repeated measures ANOVA test or the Mann-Whitney test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Effector and memory differentiation of TBEV-specific CD8 T cells.

<p>(A) Flow plots show HLA-A2 NS3 ILL⁺ CD8 T cells in one representative infected donor at day 0, 7, 21 and 90 after hospitalization (left panel), NS3 ILL-specific cells over time in five donors (right panel). (B) Flow plots representing the phenotype of TBEV-specific CD8 T cells. Plots are gated on total CD8 T cells at day 7 after hospitalization from one infected patient. (C) Results from the longitudinal phenotypic analysis of HLA-A2 NS3 ILL⁺ CD8 T cells present in the blood of five HLA-A2 patients. Bar plots show the median and 10–90th percentiles at day 7, 21 or 90 after hospitalization of CD45RA, CCR7, CD57, granzyme B, perforin and PD-1. CD27 is presented as mean fluorescence intensity (MFI). (D) Kinetics of HLA-A2 ILL-specific CD8 T cells. Bar chart represents the subset distribution of CCR7-, CD45RA-, CD57- and PD-1- expressing cells. Statistical analysis was performed using the non-parametric repeated measures ANOVA test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

TBEV peptides predicted by the NET-CTL algorithm.

<p>TBEV peptides predicted by the NET-CTL algorithm.</p