MOESM4 of Validation of an IFNÎł/IL2 FluoroSpot assay for clinical trial monitoring

Additional file 4: Table S4. Diagnostic specificity and sensitivity of the EBV-specific IFNÎł/IL2 FluoroSpot

table_1_Impaired Immune Response to Primary but Not to Booster Vaccination Against Hepatitis B in Older Adults.docx

<p>Many current vaccines are less immunogenic and less effective in elderly compared to younger adults due to age-related changes of the immune system. Most vaccines utilized in the elderly contain antigens, which the target population has had previous contact with due to previous vaccination or infection. Therefore, most studies investigating vaccine-induced immune responses in the elderly do not analyze responses to neo-antigens but rather booster responses. However, age-related differences in the immune response could differentially affect primary versus recall responses. We therefore investigated the impact of age on primary and recall antibody responses following hepatitis B vaccination in young and older adults. Focused gene expression profiling was performed before and 1 day after the vaccination in order to identify gene signatures predicting antibody responses. Young (20–40 years; n = 24) and elderly (>60 years; n = 17) healthy volunteers received either a primary series (no prior vaccination) or a single booster shot (documented primary vaccination more than 10 years ago). Antibody titers were determined at days 0, 7, and 28, as well as 6 months after the vaccination. After primary vaccination, antibody responses were lower and delayed in the elderly compared to young adults. Non-responders after the three-dose primary series were only observed in the elderly group. Maximum antibody concentrations after booster vaccination were similar in both age groups. Focused gene expression profiling identified 29 transcripts that correlated with age at baseline and clustered in a network centered around type I interferons and pro-inflammatory cytokines. In addition, smaller 8- and 6-gene signatures were identified at baseline that associated with vaccine responsiveness during primary and booster vaccination, respectively. When evaluating the kinetic changes in gene expression profiles before and after primary vaccination, a 33-gene signature, dominated by IFN-signaling, pro-inflammatory cytokines, inflammasome components, and immune cell subset markers, was uncovered that was associated with vaccine responsiveness. By contrast, no such transcripts were identified during booster vaccination. Our results document that primary differs from booster vaccination in old age, in regard to antibody responses as well as at the level of gene signatures.</p>Clinical Trial Registration<p>www.clinicaltrialsregister.eu, this trial was registered at the EU Clinical Trial Register (EU-CTR) with the EUDRACT-Nr. 2013-002589-38.</p

Overnight resting does not alter the total number of epitope specific T cells.

<p>(A) Frequencies (left panel) and functionality (right panel) of HIV and CMV specific CD8 T cells were determined by ICS. Data indicate numbers of functional T cells as% of total CD8 T cells. Pie charts show the relative contribution of each functional subpopulation within the total CD8 T-cell response according to their functionality. (B) Frequencies of HIV and CMV specific CD8 T cells determined by multimer staining. Data indicate numbers of multimer-positive T cells as% of total CD8 T cells. (A) and (B): data from five HIV (n = 3) or CMV (n = 2) seropositive individuals are shown; median indicated by black line. (C) Comparison of T cell ICS and multimer staining. Bar charts show frequencies of functional epitope specific CD8 T cells determined by ICS calculated as percentage of corresponding multimer-positive T cells. Representative results of two individuals with known epitope specificities (HLA-B8 restricted HIV Nef and HLA-A2 restricted CMV IE1, respectively) are shown. All analyses were performed on not rested and rested PBMC as indicated.</p

Overnight resting heightens sensitivity to antigens.

<p>(A) Bar charts indicate frequencies of bi- and mono-functional (blue: IFN-γ+/MIP-1β+; grey: MIP-1β+) HIV Nef specific CD8 T cells for a given peptide concentration (0.125–4 µg/ml) as percentage of total CD8 T cells. Cells were tested without (upper panel) or with (lower panel) overnight resting. (B) Pie charts show the functional profiles of HIV Nef specific CD8 T cells for a given peptide concentration. Cells were tested without (upper panel) or with (lower panel) overnight resting. (A) and (B) one representative experiment is shown. (C) Relative mean fluorescent intensity (rMFI) of IFN-γ staining of HIV Nef specific CD8 T cells of eight individuals normalized to rMFI of total CD3 T cells. All analyses were performed on not rested and rested PBMC as indicated. (** p<0.005, Wilcoxon matched pairs test; median indicated by black line).</p

Overnight resting reduces post-thaw viability of PBMC.

<p>(A) Flow cytometric analysis of lymphocyte viability compared between not rested and rested cells in a cohort of 21 individuals (** p<0.005, Wilcoxon matched pairs test; median indicated by black line). (B) Flow cytometric live/dead (NIR-/NIR+) discrimination and costaining with Annexin V for resolution between dead (NIR+) and apoptotic (NIR-/Annexin V+) cells in not rested and rested PBMC. NIR: near-infrared.</p

Antibodies used for ICS of whole blood.

<p>Antibodies used for ICS of whole blood.</p

Overnight resting increases the magnitude of antiviral T-cell responses detectable by ICS.

<p>(A) Comparison of total numbers of functional CD8 (left panel) and CD4 (right panel) T cells specific for HIV, HBV, HCV and EBV in a cohort of 21 individuals. (B) Single marker expression of antiviral CD8 (left panel) and CD4 (right panel) T cells. All analyses were performed on not rested and rested PBMC as indicated. Background values as determined in unstimulated controls are subtracted and a predefined threshold on subpopulation level is applied before calculating the total response and amount of single cytokines (see materials and methods). (*** p≤0.001; ** p≤0.05; * p≤0.05, Wilcoxon matched pairs test; median indicated by black line).</p

Overnight resting increases functionality of antiviral T cells.

<p>(A) HIV Nef specific CD8 (upper panel) and CD4 (lower panel) T cells were determined by ICS. Unstimulated PBMC served as negative controls (neg ctrl). Dot plots show functional T cells with numbers as% of total CD8 and CD4 T cells, respectively. Pie charts show the relative contribution of each T-cell subpopulation within the total HIV Nef specific T-cell response according to their functionality (mono- to poly-functional). One representative experiment is shown. (B) Pie charts represent the relative functional composition of total HIV, HBV, HCV and EBV specific CD8 (upper panel) and CD4 (lower panel) T-cell responses in a cohort of 21 individuals. All analyses were performed on not rested and rested PBMC as indicated.</p

Antibodies used for ICS of PBMC.

<p>Antibodies used for ICS of PBMC.</p

Resting effect is not mediated by antigen presenting cells.

<p>(A) Autologous EBV-transformed B-lymphoblastoid cell lines were used as antigen presenting cells to stimulate antigen specific T cells of an EBV seropositive subject. PBMC without the addition of EBV-transformed B-lymphoblastoid cell lines provided the negative controls used for background subtraction. (B) CD3 T cells of a CMV seropositive subject were isolated by magnetic cell sorting. After cryopreservation, CD3 T cells were stimulated directly or after overnight rest with a pool of overlapping peptides corresponding to the CMV-IE-1 protein. (A) and (B) IFNγ, IL2 and MIP1β production was determined by ICS directly or after an overnight resting. Frequencies (bar charts) and functional composition (pie charts) of EBV (A) and CMV (B) specific CD8 T cells are shown. CD8 T-cell subpopulations are depicted according to their functionality (three functions: green; two functions: blue; monofunctional cells: grey). One representative experiment is shown.</p