Donor details and serology.

<p>ND – not detected.</p><p>Phenotype 1 – CD27+ CD28+ perforin− granzyme B−.</p><p>Phenotype 2 – CD27+ CD28+ perforin− granzyme B+.</p><p>Phenotype 3 – CD27− CD28− perforin+ granzyme B+.</p

Expression of inhibitory receptors is associated with increased ILI-specific CD8 T cell frequency but impaired functionality.

<p>A2-ILI-tetramer+ CD8 T cells were co-stained with anti-PD-1 and 2B4, and analysed using flow cytometry. (a) Representative donors with each phenotype are shown. Numbers represent frequency of events as a percentage of CD3+CD8+ lymphocytes. (b) Non-linear regression and Spearman's rank correlation were used to show the association between the frequency of IFN-γ producing A2-ILI+ cells and their frequency of PD-1 and 2B4 co-expression. (c) Non-linear regression and Spearman's rank correlation were used to show the association between the frequency of A2-ILI tetramer+ CD8 T cells and their expression of both PD-1 and 2B4. P-values for Spearman's rank correlation are shown.</p

Summary of demographic data of HLA-A*0201+ subjects.

<p>Summary of demographic data of HLA-A*0201+ subjects.</p

ILI-specific CD8 T cells are broadly reactive, recognizing homologous epitopes conserved between alpha- and gamma-herpesviruses.

<p>Homologous epitopes to ILIEGIFFV were identified by sequence similarity in HSV-1, HSV-2 and EBV. (a) The capacity of each epitope to stimulate cytokine production was measured by <i>in vitro</i> peptide stimulation. Two representative subjects are shown with their serostatus with respect to the herpesviruses tested. Numbers represent the percentage of CD3+CD8+ lymphocytes. (b) PBMCs from HLA-A*0201+ individuals with detectable ILI-specific responses underwent <i>in vitro</i> stimulation with each epitope at a concentration of 10 µg/ml followed by staining with anti-CD3, CD8, IFN-γ and TNF-α. The frequency of cytokine producing cells stimulated by the epitopes from VZV (black), HSV-1 (light blue), EBV (red), and HSV-2 (green) as a percentage of maximal cytokine producing cell frequency is shown. (c) The ability of each epitope to induce cytokine production was assessed by titration of stimulating peptide concentrations. One representative subject (subject 118, seropositive for all herpesviruses tested) is shown. The frequency of cytokine producing cells stimulated by the epitopes from VZV (black), HSV-1 (light blue), EBV (red), and HSV-2 (green) as a percentage of maximal cytokine producing cell frequency is shown.</p

A2-ILI tetramer+ CD8 T cells display distinct phenotypic patterns between individuals.

<p>A2-ILI tetramer+ CD8 T cells were co-stained with anti-CD45RA, CCR7, CD27, CD28, granzyme B, granzyme K, perforin, and Ki-67. Phenotypic markers were analysed by flow cytometry. (a) Subjects were categorized according to the combination of phenotypic markers expressed. One representative donor of each phenotype is shown. Numbers represent percentage of CD3+CD8+ lymphocytes. (b) The frequency of A2-ILI+ CD8 T cells in every subject expressing each marker is summarized. The medians are shown and p-values derived from Student's t-test. (c) PBMCs were collected at baseline (day 0) and one month or six months later. A2-ILI tetramer+ CD8 T cells were co-stained with anti-CD27, CD28, granzyme B, and perforin. Phenotypic markers were analysed by flow cytometry. Representative plots from one subject of each phenotype are shown.</p

ILI-specific CD8 T cells display functional impairment associated with their phenotype.

<p>(a) PBMCs were stimulated with ILIEGIFFV peptide <i>in vitro</i> for 6 hours then co-stained for intracellular IFN-γ and IL-2. Numbers represent frequency as a percentage of CD8+ lymphocytes. The frequency of (b) IFN-γ+ cells as a proportion of A2-ILI tetramer+ cells and (c) IL-2 producing cells as a percentage of IFN-γ+ CD8 T cells are shown. P-values are derived from Student's t-test. (d) PBMCs from HLA-A*0201+ volunteers were stained with CFSE and stimulated with ILIEGIFFV peptide <i>in vitro</i> for 6 days. A2-ILI tetramer+ cells were then analyzed by flow cytometry. Numbers represent frequency as a percentage of CD8+ lymphocytes. Two representative donors are shown.</p