Journal: The Journal of Clinical Investigation

Article Title: Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals

doi: 10.1172/JCI152379

Figure Lengend Snippet: (A) Schematic representation of the workflow for the direct peptide stimulation of whole peripheral blood and the subsequent detection of cytokine secretion compared with a standard IFN-γ ELISPOT assay. (B) Six healthy individuals were vaccinated with 2 doses of BNT162b2 according to the recommended schedule (21 days apart), and whole-blood samples were longitudinally analyzed 7, 10, 20, and 30 days after each dose. The collected whole blood was either directly stimulated for 16 hours with peptide pools specific for the spike protein (red or black line) or NP (red or black shaded area), or immediately processed with Ficoll density gradient centrifugation to isolate PBMCs. A standard IFN-γ ELISPOT assay using the SpG- or NP-specific peptide pools was then set up using the freshly isolated PBMCs. The quantity of secreted IFN-γ in stimulated whole blood (red line) was compared with the frequency of peptide-reactive PBMCs quantified by IFN-γ ELISPOT (black line). (C) The levels of secreted IL-2 (blue line) in whole blood stimulated with the SpG peptide pool were compared with the amount of IFN-γ detected. (D) Linear regression analysis of the concentrations of IFN-γ and IL-2 in SpG-specific peptide pool–stimulated whole blood and the corresponding frequency of spike-specific PBMCs (n = 6; 48 samples). Dotted lines denote the 95% CI.

Article Snippet: The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase (streptavidin-AP) and developed using the KPL BCIP/NBT phosphatase substrate (Seracare Life Sciences).

Techniques: Enzyme-linked Immunospot, Gradient Centrifugation, Isolation

Journal: The Journal of Clinical Investigation

Article Title: Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals

doi: 10.1172/JCI152379

Figure Lengend Snippet: (A) The top matrix shows the significance of the correlation, and the Spearman’s correlation coefficient is shown in the matrix below (n = 6; 24 samples). (B) Linear regression analysis of the concentrations of IFN-γ and IL-2 in SpG peptide pool–stimulated whole blood and the corresponding frequency of SpG-reactive T cells in cryopreserved PBMCs quantified by either IFN-γ ELISPOT or AIM assay (n = 6; 24 samples). Dotted lines denote the 95% CI.

Article Snippet: The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase (streptavidin-AP) and developed using the KPL BCIP/NBT phosphatase substrate (Seracare Life Sciences).

Techniques: Enzyme-linked Immunospot

Journal: The Journal of Clinical Investigation

Article Title: Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals

doi: 10.1172/JCI152379

Figure Lengend Snippet: (A) Schematic representation of the 7 spike-specific peptide pools containing 15 mer overlapping peptides spanning the entire spike protein. Pools 1–4 contain peptides from the signal peptide and the S1 chain, whereas pools 5 and 6 encompass the S2 chain together with the transmembrane and cytoplasmic domains. (B) Plots show the longitudinal evaluation of spike-specific T cell responses (pools 1–7) by quantification of IFN-γ (left) or IL-2 (middle) in peptide-stimulated whole blood, or by IFN-γ ELISPOT (right) in 2 representative vaccinees. (C) Heatmap shows the spike-specific T cell responses quantified longitudinally in all vaccinees (n = 6) using the 3 different assays described above. “X” denotes time points that were untested.

Article Snippet: The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase (streptavidin-AP) and developed using the KPL BCIP/NBT phosphatase substrate (Seracare Life Sciences).

Techniques: Enzyme-linked Immunospot

Journal: The Journal of Clinical Investigation

Article Title: Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals

doi: 10.1172/JCI152379

Figure Lengend Snippet: (A) Schematic representation of the individual 15 mer overlapping peptides contained in the SpG peptide pool. (B) Linear regression analysis of the T cell response against the SpG peptide pool and the total spike protein (pools 1–7) as evaluated by ELISPOT (left) or by the quantification of IFN-γ (middle) or IL-2 (right) in peptide-stimulated whole blood (n = 6; 42 samples). (C) The SpG peptide pool–specific T cell response quantified by each assay is expressed as a fraction of the total spike protein T cell response observed (n = 6; 42 samples). Bars indicate the mean.

Article Snippet: The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase (streptavidin-AP) and developed using the KPL BCIP/NBT phosphatase substrate (Seracare Life Sciences).

Techniques: Enzyme-linked Immunospot

Journal: The Journal of Clinical Investigation

Article Title: Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals

doi: 10.1172/JCI152379

Figure Lengend Snippet: Linear regression analysis of IFN-γ and IL-2 concentrations in SpG peptide pool–stimulated whole blood and the corresponding frequency of spike-specific T cells quantified by IFN-γ ELISPOT in vaccinated individuals (gray; n = 30; 30 samples) and in asymptomatic (Asymp., green; n = 51; 51 samples) and symptomatic (Symp., red; n = 38; 62 samples) COVID-19 patients sampled 3 months or more after the boost vaccination dose or viral clearance. Dotted lines indicate the 95% CI. SFU, spot-forming units.

Article Snippet: The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase (streptavidin-AP) and developed using the KPL BCIP/NBT phosphatase substrate (Seracare Life Sciences).

Techniques: Enzyme-linked Immunospot

Journal: The Journal of Clinical Investigation

Article Title: Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals

doi: 10.1172/JCI152379

Figure Lengend Snippet: (A) SARS-CoV-2 spike–specific T cell responses in vaccinated individuals (n = 112; 201 samples) and in convalescent asymptomatic (n = 62; 62 samples) and symptomatic (n = 68; 115 samples) COVID-19 patients were longitudinally quantified by measuring IFN-γ secretion in whole blood after SpG peptide pool stimulation. Cross-reactive SARS-CoV-2 spike–specific T cells were also quantified in whole blood from individuals who were infected with SARS-CoV-1 eighteen years ago (n = 12; 12 samples). The responses of individuals before receiving BNT162b2 vaccination are shown for reference. Pie chart shows the number of peptides in the SpG peptide pool that are conserved or unique between SARS-CoV-2 and SARS-CoV-1. The sampling timespan (highlighted in yellow) is shown, and the number of samples analyzed at each time point is indicated in parentheses. Dashed lines denote the detection cutoff for the measured cytokines. D~14, day ~14. (B) Quantities of secreted IFN-γ (red) and IL-2 (blue) in SpG peptide pool–stimulated whole blood from vaccinees and COVID-19 patients sampled 2–3 months after a boost vaccination dose (Vacc.) or viral clearance. The bars indicate the median value for each group, and the dashed lines indicate the detection cutoff for the measured cytokines. Significant differences were analyzed and are displayed as above. (C) Longitudinal dynamics of secreted IFN-γ (red) and IL-2 (blue) in SpG peptide pool–stimulated whole blood from vaccinees and patients with COVID-19. Dashed lines indicate the detection cutoff for the measured cytokines. Significant differences in each group were analyzed by 1-way ANOVA, and the P value (adjusted for multiple comparisons) are shown. NS = P > 0.05. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Article Snippet: The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase (streptavidin-AP) and developed using the KPL BCIP/NBT phosphatase substrate (Seracare Life Sciences).

Techniques: Infection, Sampling

Journal: The Journal of Clinical Investigation

Article Title: Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals

doi: 10.1172/JCI152379

Figure Lengend Snippet: (A) SARS-CoV-2 spike–specific T cell responses were evaluated by SpG peptide pool stimulation of whole blood from vaccinated individuals (n = 27) 2 weeks (green circle) and 3 months (red circle) after the boost vaccination dose. The secreted IFN-γ and IL-2 concentrations are shown. ****P < ≤ 0.0001, by Wilcoxon matched-pairs signed-rank test. (B) Bivariate dot plots of secreted IFN-γ and IL-2 concentrations. Arrows connect paired individuals analyzed on day ~14 and day 90. Dashed lines indicate the detection cutoff for the measured cytokines.

Article Snippet: The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase (streptavidin-AP) and developed using the KPL BCIP/NBT phosphatase substrate (Seracare Life Sciences).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Rapid measurement of SARS-CoV-2 spike T cells in whole blood from vaccinated and naturally infected individuals

doi: 10.1172/JCI152379

Figure Lengend Snippet: Linear regression analysis of IFN-γ and IL-2 concentrations in SpG peptide pool–stimulated whole blood and the corresponding SARS-CoV-2 neutralizing capacity of serum from vaccinated individuals (n = 91; 112 samples) and from asymptomatic (n = 62; 62 samples) and symptomatic (n = 64; 107 samples) COVID-19 patients sampled at all time points available. Dotted lines indicate the 95% CI. sVNT, surrogate virus neutralization test.

Article Snippet: The plates were then incubated with a human biotinylated IFN-γ detection antibody, followed by streptavidin–alkaline phosphatase (streptavidin-AP) and developed using the KPL BCIP/NBT phosphatase substrate (Seracare Life Sciences).

Techniques: Neutralization

Journal: JCI Insight

Article Title: Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy

doi: 10.1172/jci.insight.122467

Figure Lengend Snippet: (A) Gating strategy used in flow cytometry–based sort for CD3+PD-1+ and/or CD134+ TILs. (B–D) ELISPOT assays measuring IFN-γ secretion of microwell cultures upon coculture with target cells. (B) Following expansion, pools of 2 cultures were tested against autologous DCs pulsed with 2 peptide pools (PP), indicated by symbols. (C) Cultures from the reactive pools were tested separately against autologous DCs pulsed with all suspected peptide pools. (D) IFN-γ ELISPOT and CD137 flow cytometry analysis showing reactivity of the TIL cultures following coculture with autologous DCs pulsed with single 25mer peptides from each peptide pool. (E) Allogeneic T cells retrovirally transduced with neoantigen-reactive TCRs cocultured with autologous DCs pulsed with serially diluted mutated and WT 25mer peptides. ‘>’ denotes greater than 500 spots. All data are representative of at least 3 independent experiments except in A.

Article Snippet: After 16–20 hours of coculture, cells were harvested from the ELISPOT plates into a standard 96-well round-bottom plate for flow cytometry analysis, and then the ELISPOT plates were washed 5 times with PBS plus 0.05% Tween 20 (PBS-T), and incubated for 2 hours at room temperature with 100 μl/well of a 0.22-μm-filtered 1 μg/ml biotinylated anti–human IFN-γ detection antibody solution (Mabtech, clone 7-B6-1, diluted in 1× PBS supplemented with 0.5% FBS).

Techniques: Flow Cytometry, Enzyme-linked Immunospot, Transduction

Journal: JCI Insight

Article Title: Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy

doi: 10.1172/jci.insight.122467

Figure Lengend Snippet: (A and B) Pools of TIL cultures incubated with DCs pulsed with pooled peptide pools (PP), indicated by symbols. (A) Summary of TIL culture pools showing secretion of IFN-γ in ELISPOT. (B) Cells from ELISPOT coculture wells were collected, labeled, and single cells expressing T cell activation markers were sorted (as shown in the bottom right panel) into a 96-well PCR plate containing lysis buffer and PCR primers for TCRα and -β. (C) CDR3β of the sequenced culture well. Bolded are the origin of the reactive TCR, based on functional assays done with the individual cultures (data not shown). (D) Expanded cultures from the indicated TIL microwells coincubated with autologous DCs pulsed with single peptides from the pools that the cultures showed recognition against in previous experiments, representative of at least 2 independent experiments. ‘>’ denotes greater than 500 spots.

Article Snippet: After 16–20 hours of coculture, cells were harvested from the ELISPOT plates into a standard 96-well round-bottom plate for flow cytometry analysis, and then the ELISPOT plates were washed 5 times with PBS plus 0.05% Tween 20 (PBS-T), and incubated for 2 hours at room temperature with 100 μl/well of a 0.22-μm-filtered 1 μg/ml biotinylated anti–human IFN-γ detection antibody solution (Mabtech, clone 7-B6-1, diluted in 1× PBS supplemented with 0.5% FBS).

Techniques: Incubation, Enzyme-linked Immunospot, Labeling, Expressing, Activation Assay, Lysis, Functional Assay

Journal: JCI Insight

Article Title: Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy

doi: 10.1172/jci.insight.122467

Figure Lengend Snippet: TILs from tumor digest were sorted and expanded based on the expression of PD-1 and/or CD134. (A) Cells from 2 cultures were combined and cocultured with DCs that were pulsed with pools of mutated peptides. Showing cultures displayed enhanced IFN-γ secretion against peptide pools 9 and 10 (pool 9 encompassing TP53 mutated peptide) in (B) IFN-γ ELISPOT assay at 16 hours. Following expansion, 2 × 104 cells from individual TIL cultures that showed specific reactivity against peptide pool 9 (not shown) were cocultured with 1 × 105 DCs pulsed with single peptides present in the pool. (C and D) TCRs targeting TP53G245S were sequenced, synthesized, and virally delivered into allogeneic PBMCs to assess reactivity and specificity. (C) TCR-transduced cells were coincubated with COS7 cells that were transfected with plasmids encoding the patient’s HLA class II and pulsed with mutated 25mers. (D) Cells were coincubated with autologous DCs pulsed with serially diluted TP53G245S and WT peptides. ‘>’ denotes greater than 500 spots. All data are representative of at least 2 independent experiments except in A.

Article Snippet: After 16–20 hours of coculture, cells were harvested from the ELISPOT plates into a standard 96-well round-bottom plate for flow cytometry analysis, and then the ELISPOT plates were washed 5 times with PBS plus 0.05% Tween 20 (PBS-T), and incubated for 2 hours at room temperature with 100 μl/well of a 0.22-μm-filtered 1 μg/ml biotinylated anti–human IFN-γ detection antibody solution (Mabtech, clone 7-B6-1, diluted in 1× PBS supplemented with 0.5% FBS).

Techniques: Expressing, Enzyme-linked Immunospot, Synthesized, Transfection

Journal: JCI Insight

Article Title: Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy

doi: 10.1172/jci.insight.122467

Figure Lengend Snippet: CD3+PD-1+ and/or CD134+ TILs were sorted, expanded at 3 cells/well, and cultures that grew were tested. (A) TIL microwell culture that showed recognition against DCs pulsed with pooled peptide pools (PP) were expanded and IFN-γ secretion was assessed following coculture for 16–20 hours with DCs pulsed with single peptide pools, and (B) single peptides from PP1. (C and D) The functionality of autologous PBMCs virally transduced with the TCR isolated from neoantigen-reactive culture was measured following incubation with (C) DCs liposomally transfected with full-length RNA encoding for KRASWT, KRASG12V, and KRASG12D, and (D) DCs loaded with supernatant from lysed cell lines that underwent 5 cycles of freezing and thawing at 1:5:10 ratio (T cells/DCs/cell lines). (E) Autologous DCs pulsed with the mutated peptide were incubated with HLA-blocking antibodies for 2 hours prior to the addition of the PBMCs expressing the TCR. (F) Effector cells expressing the TCRs were incubated with DCs (pulsed with the mutated peptide) from donors matched at one of the DRB1 alleles or with DCs from a complete DRB1 mismatch. ‘>’ denotes greater than 500 spots. All data are representative of at least 3 independent experiments.

Article Snippet: After 16–20 hours of coculture, cells were harvested from the ELISPOT plates into a standard 96-well round-bottom plate for flow cytometry analysis, and then the ELISPOT plates were washed 5 times with PBS plus 0.05% Tween 20 (PBS-T), and incubated for 2 hours at room temperature with 100 μl/well of a 0.22-μm-filtered 1 μg/ml biotinylated anti–human IFN-γ detection antibody solution (Mabtech, clone 7-B6-1, diluted in 1× PBS supplemented with 0.5% FBS).

Techniques: Transduction, Isolation, Incubation, Transfection, Blocking Assay, Expressing

Journal: BMC Infectious Diseases

Article Title: Potential of novel Mycobacterium tuberculosis infection phase-dependent antigens in the diagnosis of TB disease in a high burden setting

doi: 10.1186/1471-2334-12-10

Figure Lengend Snippet: IFN-γ levels (pg/ml) elicited by classical TB antigens in 23 TB patients (TB) and 20 household contacts (HHC) . Responses in TB cases are indicated by open symbols and those in household contacts by closed symbols. Error bars represent the median. Ag85A/B = Ag85A and Ag85B tested together, HSP = heat shock protein, PHA = Phytohaemagglutinin.

Article Snippet: Plates were then washed and 1 μg/ml (100 μl) of the biotinylated mouse-anti-human IFN-γ detection antibody (BD PharmingenTM) added.

Techniques:

Journal: BMC Infectious Diseases

Article Title: Potential of novel Mycobacterium tuberculosis infection phase-dependent antigens in the diagnosis of TB disease in a high burden setting

doi: 10.1186/1471-2334-12-10

Figure Lengend Snippet: Number of inclusions of antigens into the 10 general discriminant analysis models that most accurately predicted the presence or absence of TB disease . PHA was evaluated in all study participants as a positive control, and appeared in one of the models because IFN-γ data was analyzed in a blinded manner.

Article Snippet: Plates were then washed and 1 μg/ml (100 μl) of the biotinylated mouse-anti-human IFN-γ detection antibody (BD PharmingenTM) added.

Techniques: Positive Control