cmv pp65  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

doi: 10.1101/2025.07.15.664847

Figure Lengend Snippet: ( A ) Schematic depicting the AIM assay procedure and protocol variations that were tested: (1) presence/absence of human serum, (2) use of whole blood, or fresh or frozen PBMCs, (3) stimulation with peptide, whole protein or other antigens; (4), stimulation time; (5) AIM marker combinations and (6) and data analysis strategy. ( B-E ) Replicate AIM assays were performed on cryopreserved PBMCs from 5 donors, with 4-8 technical replicates per donor spread across 2-4 independent experiments. PBMCs were stimulated for 20 h with CMV pp65 peptides and AIM responses were measured by flow cytometry as CD4 + or CD8 + T cells co-expressing the indicated AIMs. (B and C) Representative dot plots showing AIM + cells after gating on CD4 + (B) or CD8 + (C) T cells. (D and E) paired raw AIM + frequencies among CD4 + (D) and CD8 + (E) T cells. Percentage coefficients of variation (CV) are displayed above the responses for each donor. Each line represents the paired unstimulated control and pp65-stimulated AIM signal from a single technical replicate within one donor. ( F and G ) The source(s) of variance contributing to variability in pp65-stimulated CD4 + (F) and CD8 + (G) AIM + cell frequencies were calculated after subtracting the corresponding unstimulated AIM + frequencies. A mixed effects model with donor, experiment (technical replicate) and operator (the individual performing the AIM assay) as random effects was used. Note that for CD137 + /CD69 + (CD4 + ) and CD107a + /CD137 + (CD8 + ) data, the mixed effects model failed to converge and variance components could not be calculated. Graphs show variance components expressed as percentages of total variance.

Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

Techniques: Marker, Flow Cytometry, Expressing, Control

Journal: bioRxiv

Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

doi: 10.1101/2025.07.15.664847

Figure Lengend Snippet: ( A ) Re-analysis of previously published AIM assay data from SARS-CoV-2 Spike peptide-stimulated PBMCs from solid organ transplant recipients (n = 33) vaccinated with three doses of a COVID-19 mRNA vaccine (PREVENT cohort) , or ( B ) new analysis of CMV pp65-stimulated PBMCs from 6 healthy donors assayed across 3 independent experiments at each of 4 distinct research centres in the CAN-ASC cohort (n = 52 total). Correlation and linear regression analyses of unstimulated AIM + frequencies with raw antigen-stimulated AIM + frequencies (left panel), or net responses after subtracting (centre panel) or dividing by (right panel) the unstimulated condition. CD134 + /CD25 + frequencies among CD4 + T cells (upper panel) and CD137 + /CD69 + frequencies among CD8 + T cells (lower panel) are presented. Spearman ρ and p-values are shown from Spearman correlation tests. The solid line is derived from linear regression analysis. For the CAN-ASC cohort, regression and correlation analyses were performed after correcting for between-donor variation ( C ) Schematic showing the workflow of the Box-Cox transformation method of correcting for AIM + cells in the unstimulated control. Raw AIM + frequencies are transformed using the Box-Cox formula with a user-defined parameter l؝[0,1] prior to subtracting to obtain a net signal on the Box-Cox-transformed scale. Inverse Box-Cox transformation of the difference restores the AIM response to the original scale, yielding the stimulation index (SI). ( D and E ) Three statistical methods to estimate optimal values of λ are presented, seeking to minimize correlation between the unstimulated and net stimulated AIM values. Left panel: linear regression, to identify λ giving the highest probability of zero slope (̂β = 0), expressed as a posterior probability distribution for λ. Centre panel: Spearman correlation to identify the value of λ resulting in an estimated zero correlation. Right panel: likelihood profile for λ based on linear regression to estimate the probable optimal value of λ. Values were estimated for the (D) PREVENT or (E) CAN-ASC cohorts. ( F ) Analyses of the CAN-ASC cohort showing ratios of biological (between-donor) variance to total variance (intra-class correlation, ICC; green solid line) plotted as a function of λ using the Box-Cox-corrected stimulation index for CD4 + and CD8 + AIM responses to CMV pp65. ICC ratios for raw (untransformed) AIM + frequencies are indicated by the grey dotted line. ( G ) Comparison of CVs calculated between technical replicates within individual donors for raw AIM + frequencies, subtraction of or division by the unstimulated AIM + frequency, or Box-Cox-corrected SI. Each point is the CV of technical replicates from one donor, representing within-donor technical variability. P-values are shown from Dunnett’s multiple comparisons test following one-way repeated measures ANOVA. CVs were adjusted to account for the effect of a change in distribution using bootstrap estimates, where the same transformation was repeatedly applied after randomly replacing unstimulated values. ( H and I ) Re-analysis of AIM responses for (H) CD4 + and (I) CD8 + T cells in n = 40 solid organ transplant recipients from the PREVENT cohort throughout a three-dose COVID-19 mRNA vaccination schedule calculated by subtracting (left) or dividing by (centre) the AIM + frequencies in the unstimulated condition, or Box-Cox-corrected SI values (right). P-values were calculated from log 2 -transformed data using a mixed-effects model with Dunnett’s multiple comparisons test. The bolded p-values denote significant differences between timepoints detected only by analysis of Box-Cox-corrected SI values. See also Figures S2-S5 and Document S1.

Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

Techniques: Derivative Assay, Transformation Assay, Control, Comparison

Journal: bioRxiv

Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

doi: 10.1101/2025.07.15.664847

Figure Lengend Snippet: Comparisons of AIM responses using fresh whole blood, or fresh (PBMC) or cryopreserved (PBMC-C) peripheral blood mononuclear cells, with or without a 24-h delay in processing (- 24h). Cells were rested overnight and stimulated for 20 h with cytomegalovirus (CMV) pp65 peptides, CMV pp65 whole protein, or Infanrix vaccine. ( A ) Percentage viable (fixable viability dye-eFluor 780-negative) cells of total CD3 + T cells measured by flow cytometry in AIM assays. Error bars represent the standard deviation of the mean. P-values are shown for planned comparisons using Dunn’s multiple comparisons test following a Friedman test. ( B-E ) Representative flow cytometry (B and C) and quantification of stimulation index for each AIM relative to the unstimulated control (D and E) among CD4 + (B and D) and CD8 + (C and E) T cells from each cell source. ( F ) Ratio of AIM stimulation indices between CMV pp65 protein- and peptide-stimulated CD4 + T cells in relation to cell source. Positive or negative ratios indicate greater detection of AIM responses with whole protein or peptide stimulation, respectively, while confidence intervals overlapping zero indicate no significant difference between protein and peptide stimulation. (D, E and F) Error bars are 95% confidence intervals calculated on a log 2 scale. (A-F) Data from n=6 healthy donors. See also Figure S7.

Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

Techniques: Flow Cytometry, Standard Deviation, Control

Journal: bioRxiv

Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

doi: 10.1101/2025.07.15.664847

Figure Lengend Snippet: ( A ) Representative and ( B ) quantified flow cytometry data showing CD134 + /CD25 + cells among CD4 + T cells following stimulation of cryopreserved PBMCs (PBMC-C) or fresh whole blood with CMV pp65 peptides for 6, 20 or 44 h. ( C ) Representative and ( D ) quantified flow cytometry showing CD137 + /CD69 + cells among CD8 + T cells following stimulation of PBMC-C or fresh whole blood with CMV pp65 peptides for 6, 20 or 44 h. (B and D) SIs are represented as log 2 - transformed data. P-values are shown from one-sample t-tests, with AIM signals considered present when the mean log 2 -transformed SI significantly differs from zero. All data are paired samples from n = 4 healthy donors. ns, not significant (p > 0.05); *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. See also Figure S8.

Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

Techniques: Flow Cytometry, Transformation Assay

Journal: bioRxiv

Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

doi: 10.1101/2025.07.15.664847

Figure Lengend Snippet: (A) Representative flow cytometric gating strategy for FOXP3 + /HELIOS + Tregs. ( B ) Effect of stimulation with cytomegalovirus (CMV) pp65 peptides on FOXP3 + and FOXP3 + /HELIOS + cell frequencies among CD4 + T cells over time. P-values represent Tukey’s multiple comparisons test following a two-way ANOVA.( C-E ) Stimulation indices (C and E) or representative flow cytometry (D) for AIM pairs and Boolean combinations in FOXP3 + HELIOS + Tregs following stimulation of cryopreserved PBMCs with pp65 peptides, CMV pp65 whole protein, SARS-CoV-2 Spike peptides or the Infanrix combination vaccine for 20 h. Boolean AND/OR gating combinations comprise CD134/CD69, CD134/CD137 and CD137/CD69 (3xAIM); or 3xAIM plus CD154/CD69, CD134/CD154 and CD137/CD154 (6xAIM). Stimulation index was determined by normalizing pp65 peptide-stimulated AIM + frequencies to the unstimulated control using the box-cox correction method described in . The mean log 2 -transformed stimulation index is shown for each AIM. Error bars represent 95% confidence intervals of the mean. Data represent n = 9 (pp65 and Infanrix 20-h stimulations) and n = 4 (all others) healthy donors. ( F ) Flow cytometric gating strategy to evaluate CD39 + frequencies among CD134 + /CD137 + and non-CD134+/CD137+ (comprising CD134 + /CD137 - , CD134 - /CD137 - and CD134 - /CD137 + cells) FOXP3 + /HELIOS + Tregs and FOXP3/HELIOS - Tconvs. ( G ) Median percentages of CD39 + events among CD134 + /CD137 + or CD134/CD137 - CD4 + /FOXP3 + /HELIOS + Tregs or conventional FOXP3/HELIOS - T cells (Tconvs). ( H ) Positive predictive value (PPV) of CD39 (top) or CD39, CD134 and CD137 (bottom) as markers for antigen-specific Tregs (CD134 + /CD137 + /FOXP3 + /HELIOS + ) among total CD4 + T cells. ns, not significant (p > 0.05); *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. See also Figure S9.

Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

Techniques: Flow Cytometry, Control, Transformation Assay

Journal: bioRxiv

Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

doi: 10.1101/2025.07.15.664847

Figure Lengend Snippet: ( A ) Experimental design for AIM assay inter-site testing. Replicate aliquots of PBMCs from n = 6 healthy donors were assayed in three independent experiments (individual points) by four distinct Canadian research groups (coded A-D). Replicate AIM assay experiments (n = 3 per donor, per site) were performed by stimulating PBMCs (thawed independently for each replicate) for 20 h with CMV pp65 or SARS-CoV-2 Spike peptides. ( B and C ) Stimulation indices of CD134 + /CD25 + frequencies among CD4 + T cells (top) and CD137 + /CD69 + frequencies among CD8 + T cells (bottom). Figure subpanels separate individual donors. Stimulation index was determined by normalizing pp65 peptide-stimulated AIM + frequencies to the unstimulated control using the Box-Cox correction method described in . The means and standard deviations of log 2 -transformed stimulation indices are shown for each AIM. Symbol colours indicate the percent coefficient of variation (CV) within each set of three replicates at each site. The overall CV between sites is shown at the top of the panel for each donor. ( D ) Percent CV in CD4 + (top) and CD8 + (bottom) T cell AIM stimulation indices calculated between technical replicate AIM assays within each site for unstimulated, pp65- stimulated and Spike-stimulated conditions. Each point represents the CV of three technical replicates within one site; bars represent individual donors. CV means with standard deviation are shown. The dotted line represents CV = 30%, a common threshold for acceptable assay reproducibility. ( E and F ) ICCs comparing the technical variability among CD4 + (top) and CD8 + (bottom) AIM responses to (D) CMV pp65 and (E) SARS-CoV-2 Spike using data from replicate AIM assays from different sites and donors. Bars represents the ICC for each AIM as a mean across all sites. The dotted line indicates ICC = 0.5, the point at which biological and technical variability are equal. ( G and H ) Percent CV in CD4 + (top) and CD8 + T cell (bottom) AIM responses to CMV pp65 (F) and SARS-CoV-2 Spike (G) between sites, calculated after averaging three technical replicate AIM assays at each site. Each point represents one donor. Error bars are 95% confidence intervals of the mean. The dotted line represents CV = 30%, a common threshold for acceptable assay reproducibility. ( I ) ICCs comparing the calculated technical variability for replicate AIM assays between sites to biological variability between donors for CD4 + (top) and CD8 + (bottom). The dotted line indicates ICC = 0.5, the point at which biological and technical variability are equal. See also Figures S10-S14.

Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

Techniques: Control, Transformation Assay, Standard Deviation

Journal: bioRxiv

Article Title: Reproducible detection of antigen-specific T cells and Tregs via standardized and automated activation-induced marker assay workflows

doi: 10.1101/2025.07.15.664847

Figure Lengend Snippet: (A) Individuals were trained on identical gating protocols and analyzed the same data files from three technical replicates of 20-h AIM assays with CMV pp65 peptides or SARS-CoV-2 Spike peptides on healthy donor PBMCs (n = 6) collected at four distinct sites designated A-D. Raw flow cytometric data from each replicate at each site was analyzed independently by 3 different individuals. Data represent CVs between individual analysts, as a mean of the analyst CV for the three technical replicates of each donor-site combination. (B and C) An automated AIM gating pipeline was created via analysis of AIM assay data from healthy donors (n = 6) assayed in triplicate at each of four research centres. Comparative flow cytometric gating approaches for manual and automated analyses are shown for (B) CD4 + CD134 + /CD25 + and (C) CD8 + CD137 + /CD69 + T cells following a 20-h incubation of cryopreserved PBMCs with no antigen (unstimulated), CMV pp65 peptides, SARS-CoV-2 Spike peptides or CytoStim. The CytoStim condition was used by the automated software to set donor-specific AIM gates, which were then applied to other stimulation conditions for that donor. (D-F) Multi-centre CMV and SARS-CoV-2 AIM assay data were analyzed manually or using the automated gating software. A central analyst (C1) defined the gating strategy, provided instructions to the manual analysts (C2 and O1-O4), oversaw automated gating development, and was the reference for comparisons. C1, C2 and the automated software each analyzed all data from all sites, while O1-O4 each analyzed data from a single site (O1: A, O2: B, O3: C, O4: D). (D-F) Box-Cox-corrected AIM SI values are shown for (D) CMV and (E) SARS-CoV-2 Spike AIM assays. P-values were calculated via post-hoc Dunnett’s multiple comparisons test following mixed effects analysis and represent paired comparisons between each analyst and C1, and used to compute (F) the total number of comparisons for C2 and automated analyses that were significantly different from the analysis by C1 for CD4 + AIMs. Each of the 32 distinct combinations of CD4 + AIM, site and antigen (CMV or SARS-CoV-2) was considered to be one comparison. Pooled analysis of all comparisons was performed using Fisher’s exact test. (G-I) Spearman correlations of AIM SI values for C1, C2, O1-O4 (pooled) and automated analyses for CMV and for SARS-CoV-2 Spike. Comparisons are shown against (G and H) the reference C1 and (I) for each analyst against all others in a correlation matrix. (J) F1 scores calculated for C2 manual vs. C1 reference (grey) or automated vs. C1 reference (green) analysis of CMV and SARS-CoV-2 AIM assay data. Each point represents the F1 score from a unique donor-site combination, with p-values calculated via paired Wilcoxon signed-rank test after averaging F1 scores from technical replicates. ns, not significant (p > 0.05); *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. See also Figures S15-S19.

Article Snippet: Cells were incubated for 6, 20 or 44 h with media, 1.5 μg/mL PepTivator CMV pp65 (Miltenyi), 3.3 μg/mL CMV pp65 recombinant protein (Miltenyi), 1 μg/mL PepTivator SARS-CoV-2 Prot_S (Miltenyi), 1/100 Infanrix hexa (GSK) or 1/400 CytoStim (Miltenyi).

Techniques: Incubation, Software, Comparison