Journal: Cytokine

Article Title: Maintenance of CMV-specific CD8 + T cell responses and the relationship of IL-27 to IFN-? levels with aging

doi: 10.1016/j.cyto.2012.11.024

Figure Lengend Snippet: PBMCs were purified from the peripheral blood of young (age ≤ 40) and elderly (age ≥ 65) donors infected with CMV and stimulated for 6 hours with or without a CMV pp65 peptide pool in the presence of Golgi plug during the last 5 hours of stimulation. Stimulated cells were fixed and permeabilized using appropriate buffers followed by staining with antibodies to CD3, CD8, IFN-γ and TNF-α (BD Pharmingen). Stained cells were analyzed on an LSRII® flow cytometer. (A) Representative data showing CD8+ T cells producing IFN-γ and/or TNF-α in response to CMV pp65 peptides. (B–E) The frequency of CD8+ T cells producing IFN-γ and/or TNF-α (B), IFN-γ only (C), TNF-α only (D) and both IFN-γ and TNF-α (E) in response to CMV pp65 peptides in young (n = 12) and elderly people (n = 14). Bars and error bars indicate mean and standard error of mean (SEM), respectively.

Article Snippet: For determination of CD8 + T cell proliferation in response to CMV pp65 peptides, PBMCs were stained with carboxyfluorescein diacetate (CFSE, Molecular Probe, Eugene, OR) and stimulated for 7 days with or without the CMV pp65 peptide pool (Proimmune).

Techniques: Purification, Infection, Staining, Flow Cytometry

Journal: Cytokine

Article Title: Maintenance of CMV-specific CD8 + T cell responses and the relationship of IL-27 to IFN-? levels with aging

doi: 10.1016/j.cyto.2012.11.024

Figure Lengend Snippet: PBMCs were purified from the peripheral blood of young (age ≤ 40) and elderly (age ≥ 65) donors infected with CMV and stained with CFSE. Stained cells were stimulated for 7 days with or without a CMV pp65 peptide pool. Cell proliferation was analyzed on an LSRII® flow cytometer. (A) Representative data showing CD8+ T cell proliferation in response to CMV pp65 peptides. (B) The frequency of CD8+ T cells that proliferated in response to CMV pp65 peptides in young (n = 11) and elderly (n =15) people. Bars and error bars indicate mean and SEM, respectively. (C–D) Correlation of the frequencies of proliferating and cytokine producing CD8+ T cells in young and elderly people. P values were obtained using the unpaired t-test (B) and the Pearson correlation analysis (C–D).

Article Snippet: For determination of CD8 + T cell proliferation in response to CMV pp65 peptides, PBMCs were stained with carboxyfluorescein diacetate (CFSE, Molecular Probe, Eugene, OR) and stimulated for 7 days with or without the CMV pp65 peptide pool (Proimmune).

Techniques: Purification, Infection, Staining, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Long-term Survival in Glioblastoma with Cytomegalovirus pp65-targeted Vaccination

doi: 10.1158/1078-0432.CCR-16-2057

Figure Lengend Snippet: Schema of ATTAC-GM trial. Following standard of care with gross total resection (> 90%), external beam radiation (RT) and temozolomide (TMZ), patients received DI-TMZ cycle 1 (100 mg/m2/d) for 21 days of a 28-day cycle. DC vaccines consisted of 2×107 mature pp65- lysosome-associated membrane glycoprotein (LAMP) mRNA-pulsed DCs (pp65-DCs) admixed with 150 µg GM-CSF. Vaccination with pp65-DCs occurred on Day 23 ± 1 of the 28-day cycle with the first three DC vaccines administered two weeks apart. Following DI-TMZ cycle 2 and DC Vaccine-4, patients then received monthly DC vaccines administered on DI-TMZ cycle Day 23 ± 1 for a total of 10 vaccines in conjunction with monthly DI-TMZ cycles for a total of 6 to 12 cycles unless progression occurred. Patients were imaged bi-monthly without receiving any other prescribed anti-tumor therapy. For immune monitoring of pp65 responses, peripheral blood mononuclear cells were sampled at Pheresis-1 and Pheresis-2, along with blood draws just prior to vaccination with pp65-DCs.

Article Snippet: PBMCs were stimulated overnight with a pool of synthetic peptides spanning CMV pp65 (15-mers overlapping by 11 amino acids with > 95% purity), kindly provided by Dr. Robert A. Olmsted (Alphavax, Inc., Research Triangle Park, NC).

Techniques: Vaccines, Membrane

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Long-term Survival in Glioblastoma with Cytomegalovirus pp65-targeted Vaccination

doi: 10.1158/1078-0432.CCR-16-2057

Figure Lengend Snippet: Survival rates in patients receiving pp65-DCs and DI-TMZ compared to historical controls. A, PFS and B, OS of study patients (n = 11) with newly-diagnosed GBM receiving DI-TMZ conditioning and GM-CSF-containing pp65-DC vaccines compared to matched historical controls (n = 23) with newly diagnosed GBM treated with standard of care and additional therapies after disease progression. Kaplan Meier survival curves represent observed rates for DI-TMZ + pp65 DC patients who completed the predefined study therapy. Of all 11 patients, four had not progressed and were alive at the time of survival analysis (DI-TMZ + pp65-DCs median PFS = 25.3 months [CI95: 11.0-∞] vs. Historical controls median PFS = 8.0 months [CI95: 6.2–10.8], P = 0.0001; DI-TMZ + pp65-DCs median OS = 41.1 months [CI95: 21.6-∞] vs. Historical controls median OS = 19.2 months [CI95: 14.3–21.3], P = 0.0001, log-rank test).

Article Snippet: PBMCs were stimulated overnight with a pool of synthetic peptides spanning CMV pp65 (15-mers overlapping by 11 amino acids with > 95% purity), kindly provided by Dr. Robert A. Olmsted (Alphavax, Inc., Research Triangle Park, NC).

Techniques: Vaccines, Biomarker Discovery

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Long-term Survival in Glioblastoma with Cytomegalovirus pp65-targeted Vaccination

doi: 10.1158/1078-0432.CCR-16-2057

Figure Lengend Snippet: Patient pp65 ELISpot responses following DI-TMZ and sequential pp65-DC vaccination. A, pp65 antigen-specific T-cell responses as measured by IFN-γ ELISpot ex vivo. Before-and-after pp65 ELISpot following three vaccinations of pp65-DCs from Vaccine-1 to Pheresis-2 (mean ± sem spot-forming cells (SFC) per 106 PBMC) in all patients (n = 11) are shown after stimulation with 138 15-mer peptides overlapping by 11 amino acids spanning the entire pp65 gene (P = 0.019 Wilcoxon signed rank). B, Kinetics of pp65 ELISpot throughout continuous pp65-DC vaccination and intervening DI-TMZ cycles. Timing of DI-TMZ cycles are shown as detached lines. C, Fold changes in functional pp65 ELISpot from baseline pp65 reactivity prior to Vaccine-1. Fold increases stratified by patient OS > 40 months (n = 6) and OS < 40 months (n = 5). Post Vaccine-1: mean 1.51 vs. 3.75 (P = 0.031), Post Vaccine-2: mean 2.20 vs. 5.14 (P = 0.031), Post Vaccine-3: mean 3.45 vs. 9.79 (P = 0.031 Wilcoxon signed rank). ELISpot 0 values normalized to [0 + 1] for calculation of fold change from baseline.

Article Snippet: PBMCs were stimulated overnight with a pool of synthetic peptides spanning CMV pp65 (15-mers overlapping by 11 amino acids with > 95% purity), kindly provided by Dr. Robert A. Olmsted (Alphavax, Inc., Research Triangle Park, NC).

Techniques: Enzyme-linked Immunospot, Ex Vivo, Functional Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Long-term Survival in Glioblastoma with Cytomegalovirus pp65-targeted Vaccination

doi: 10.1158/1078-0432.CCR-16-2057

Figure Lengend Snippet: MRI changes in long-term survivors with sequential pp65-DC vaccination. A, Sequential MRI scans (FLAIR and T1-weighted with contrast) of four long term survivors receiving pp65-DCs and DI-TMZ (Patient 2, Patient 3, Patient 4, and Patient 5). Repeat MRI scans demonstrate steadily decreasing FLAIR hyperintensity and stable or decreasing contrast enhancement with collapse of the resection cavity. B, Satellite FLAIR hyperintense lesions appearing after several vaccinations with pp65-DCs in two patients with prolonged OS (Patient 4 and Patient 7). These lesions were not originally present at the post-XRT/TMZ scan and were calculated to be outside the range of XRT high-dose radiation fields. Presentation of these lesions was first detected after Vaccine-4 and Vaccine-7, and their signal persisted through Vaccine-10.

Article Snippet: PBMCs were stimulated overnight with a pool of synthetic peptides spanning CMV pp65 (15-mers overlapping by 11 amino acids with > 95% purity), kindly provided by Dr. Robert A. Olmsted (Alphavax, Inc., Research Triangle Park, NC).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Long-term Survival in Glioblastoma with Cytomegalovirus pp65-targeted Vaccination

doi: 10.1158/1078-0432.CCR-16-2057

Figure Lengend Snippet: Sequential pp65 DC vaccination expands peripheral CD8+ T cells and CD8 : TReg ratios but does not affect conventional CD4+ counts or CD4 : TReg ratios. A, CD8+ T cell counts in the peripheral blood of patients decrease following DI-TMZ cycle 1 from Pheresis-1 to Vaccine-1 (P = 0.020) and steadily increase with sequential vaccination of pp65-DCs from Vaccine-1 to Vaccine-3 (P = 0.012). B, CD8 : TReg ratios steadily increase following sequential pp65-DCs (Vaccine-1 to Vaccine-3, P = 0.004) C, Conventional CD4+ T cell counts in the peripheral blood of patients dramatically diminish following DI-TMZ cycle 1 (P = 0.037) and do not increase following sequential pp65 DC vaccination. D, CD4 : TReg ratios decrease following DI-TMZ cycle 1 (P = 0.002) but are not affected by sequential pp65-DCs (A-D, mean ± sem, Wilcoxon signed rank).

Article Snippet: PBMCs were stimulated overnight with a pool of synthetic peptides spanning CMV pp65 (15-mers overlapping by 11 amino acids with > 95% purity), kindly provided by Dr. Robert A. Olmsted (Alphavax, Inc., Research Triangle Park, NC).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Reduced Ebola vaccine responses in CMV + young adults is associated with expansion of CD57 + KLRG1 + T cells

doi: 10.1084/jem.20200004

Figure Lengend Snippet: Increased frequencies of antigen-specific T cells express CD57 and KLRG1 in CMV + young adults. (A) Expression of AIMs OX40 and CD25 on CD4 + and CD8 + T cells in PBMCs that were unstimulated (unstim) or stimulated with CMV pp65, Ebola GP, or Staphylococcal enterotoxin B (SEB). (B and C) Frequency of OX40 + CD25 + cells in CD4 + T cells (B) and CD8 + T cells (C) in CMV − ( n = 8) and CMV + ( n = 8) individuals. Mann–Whitney analyses. (D) Frequency of CD57 + KLRG1 + cells within GP-specific (OX40 + CD25 + ) CD4 + and CD8 + T cells in CMV − ( n = 8) and CMV + ( n = 8) UK adults at M+7. Mann–Whitney analyses between CMV − and CMV + groups. (E–H) Relationship between frequency of CD57 + KLRG1 + cells in CD4 + (E and G) or CD8 + (F and H) T cells and M+7 Ebola GP IFN-γ ELISPOT (E and F; CMV − n = 8, CMV + n = 8) or IL2 produced by GP-stimulated (stim) M+7 PBMCs (G and H; measured using the LEGENDplex assay; CMV − , n = 8, and CMV + , n = 6; cytokine data for two individuals not available). Spearman’s rank analyses. SFC, spot-forming cell. Medians shown for all column graphs. Error bars indicate IQRs. *, P < 0.05; ***, P < 0.001.

Article Snippet: Cells were either stimulated with 0.5 μg/ml of a pool of CMV pp65 peptides (PM-PP65-1; Cambridge Bioscience) or 2 μg/ml of Zaire Ebola GP peptides (Neoscientific).

Techniques: Expressing, MANN-WHITNEY, Enzyme-linked Immunospot, Produced

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: Distribution of screening responses for LOD, confirmatory cutpoint, and PHA minimum evaluation PBMCs from 99 normal human donors were mock stimulated or stimulated with peptide pools from AAV5, FVIII, or CMVpp65 or PHA and tested by IFN-γ ELISpot. (A) The distribution of mock responses (outliers excluded) was used to establish the LOD as the statistical 95 th percentile. (B) The pooled distribution of antigen-specific over mock response ratios (outliers excluded) was used to establish the confirmatory cutpoint as the empirical 95 th percentile. (C) The distribution of PHA responses was used to establish the PHA minimum as the empirical 1 st percentile.

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques: Enzyme-linked Immunospot

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: Intra-triplicate, intra-assay and interassay precision

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques: Intra Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: Linearity, ULOQ, and specificity evaluation (A) Using PBMCs from 3 medium to strong CMVpp65-responding donors, IFN-γ ELISpot was performed by stimulation to CMVpp65 in 3 different runs at 7 different cell densities (400,000–6,250 PBMCs/well). Each symbol indicates a different donor (●, ▲, and ▼). The linear range was defined as the range of cell densities with responses ≥LOD, through which a linear regression yields a coefficient of correlation r 2 ≥0.90. (B) Human PBMCs from 6 donors were stimulated at varying concentrations with PHA in 3 runs. The ULOQ was defined as the 95 th percentile of the highest SFU/well responses elicited that consistently showed an intra-triplicate CV ≤30% (total of 18 data points). (C) PBMCs from 6 donors were stimulated with 4 negative control peptide pools (HIV, AAV5 pool 1, AAV5 pool 2, and FVIII pool 1) along with positive (CMVpp65 and PHA) and mock controls across 4 runs. Each bar color indicates a different donor. Error bars represent the standard deviation from the mean for each donor across the 4 runs. Specificity met acceptance criteria if the SFU/well for the negative control peptide pools were

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques: Enzyme-linked Immunospot, Negative Control, Standard Deviation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: LLOQ determination

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Monitoring cell-mediated immune responses in AAV gene therapy clinical trials using a validated IFN-γ ELISpot method

doi: 10.1016/j.omtm.2021.05.012

Figure Lengend Snippet: Trending and stability of control donor PBMCs (A–E) PBMCs from a control donor were monitored during clinical sample testing for IFN-γ responses to (A) mock, (B) FVIII pool 2, (C) FVIII pool 3, (D) CMVpp65, and (E) PHA. Additional FVIII or AAV5 peptide pools were not tested since they did not generate a positive response in this control donor. Responses were analyzed for trends using Levey-Jennings plots. The blue line indicates the limit of detection (LOD, or 60 SFU/million PBMCs). The dashed black line indicates the lower limit of quantification (LLOQ, or 222 SFU/million PBMCs). The green line indicates the mean of each dataset. The red lines indicate the upper confidence limit (UCL) and lower confidence limit (LCL) of the dataset, representing 3 multiples of the standard deviation above and below the mean of all of the data points plotted, respectively. (F) PHA responses that were too numerous to count were not plotted since no numerical values could be assigned. PBMC viability remained >79% throughout.

Article Snippet: Aliquots of lyophilized human CMVpp65 peptide pool (JPT Peptide Technologies GmbH) containing 138 peptides with >70% purity were purchased.

Techniques: Control, Standard Deviation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activated integrins identify functional antigen-specific CD8 + T cells within minutes after antigen stimulation

doi: 10.1073/pnas.1720714115

Figure Lengend Snippet: Flow-cytometry assessment of antigen-specific CD8 + T cells using mICAM-1. ( A ) Time course of activated β 2 -integrin staining following incubation of whole blood (WB) without antigen (black, three donors), with SEB (gray, three donors), CMV/NLV peptide (blue, three donors), CMV/pp65 pool of overlapping peptides (light green, three donors), HIV/p17 pool of overlapping peptides (dark green, one HIV-seropositive patient), EBV/GLC peptide (purple, two donors), Flu/GIL peptide (red, two donors), or YFV/LLW peptide (orange, one vaccinated subject) for 4, 8, 16, 32, 64, 128, or 256 min. For the final 4 min of incubation, mICAM-1 was added. Data show percentages of mICAM-1 + cells among total CD8 + T cells; for the antigen-stimulated sample, background from the relevant unstimulated sample was subtracted (mean ± SEM). ( B ) mICAM-1 staining obtained from three representative donors after 8 and 64/128 min of stimulation. ( C ) Examples of mICAM-1 staining of WB from three CMV-seropositive donors with low, intermediate, and high frequencies of CMV-specific cells after 8-min stimulation with CMV/NLV peptide. ( D ) mICAM-1 staining was compared in WB cells, fresh PBMCs and frozen/thawed (fz/thw) PBMCs after 8 min of stimulation with the CMV/NLV peptide. One of two donors is shown. Numbers indicate percentage of mICAM-1 + cells among the CD8 + T cells. w/o, without.

Article Snippet: We stimulated whole blood (380 µL per test) for the indicated times at 37 °C in a water bath in 5-mL Falcon tubes (BD Biosciences) with the following peptides: 4 µg/mL CMV/NLV, 4 µg/mL EBV/GLC, 4 µg/mL Flu/GIL, 4 µg/mL YFV/LLW, 2 µg/mL PSMA/ALF, peptide pools: 2 µg/mL CMV/pp65, 2 µg/mL HIV/p17, 2 µg/mL HIV/Pol, or 4 µg/mL SEB (Sigma-Aldrich).

Techniques: Flow Cytometry, Staining, Incubation