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

Antibodies used for ICS of whole blood.

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

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

DataSheet_3_Comparable cellular and humoral immunity upon homologous and heterologous COVID-19 vaccination regimens in kidney transplant recipients.pdf

BackgroundKidney transplant recipients (KTRs) are at high risk for a severe course of coronavirus disease 2019 (COVID-19); thus, effective vaccination is critical. However, the achievement of protective immunogenicity is hampered by immunosuppressive therapies. We assessed cellular and humoral immunity and breakthrough infection rates in KTRs vaccinated with homologous and heterologous COVID-19 vaccination regimens.MethodWe performed a comparative in-depth analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific T-cell responses using multiplex Fluorospot assays and SARS-CoV-2-specific neutralizing antibodies (NAbs) between three-times homologously (n = 18) and heterologously (n = 8) vaccinated KTRs.ResultsWe detected SARS-CoV-2-reactive T cells in 100% of KTRs upon third vaccination, with comparable frequencies, T-cell expression profiles, and relative interferon γ and interleukin 2 production per single cell between homologously and heterologously vaccinated KTRs. SARS-CoV-2-specific NAb positivity rates were significantly higher in heterologously (87.5%) compared to homologously vaccinated (50.0%) KTRs (P ConclusionOur data support a more comprehensive assessment of not only humoral but also cellular SARS-CoV-2-specific immunity in KTRs to provide an in-depth understanding about the COVID-19 vaccine–induced immune response in a transplant setting.</p

DataSheet_2_Comparable cellular and humoral immunity upon homologous and heterologous COVID-19 vaccination regimens in kidney transplant recipients.pdf

BackgroundKidney transplant recipients (KTRs) are at high risk for a severe course of coronavirus disease 2019 (COVID-19); thus, effective vaccination is critical. However, the achievement of protective immunogenicity is hampered by immunosuppressive therapies. We assessed cellular and humoral immunity and breakthrough infection rates in KTRs vaccinated with homologous and heterologous COVID-19 vaccination regimens.MethodWe performed a comparative in-depth analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific T-cell responses using multiplex Fluorospot assays and SARS-CoV-2-specific neutralizing antibodies (NAbs) between three-times homologously (n = 18) and heterologously (n = 8) vaccinated KTRs.ResultsWe detected SARS-CoV-2-reactive T cells in 100% of KTRs upon third vaccination, with comparable frequencies, T-cell expression profiles, and relative interferon γ and interleukin 2 production per single cell between homologously and heterologously vaccinated KTRs. SARS-CoV-2-specific NAb positivity rates were significantly higher in heterologously (87.5%) compared to homologously vaccinated (50.0%) KTRs (P ConclusionOur data support a more comprehensive assessment of not only humoral but also cellular SARS-CoV-2-specific immunity in KTRs to provide an in-depth understanding about the COVID-19 vaccine–induced immune response in a transplant setting.</p