cd8 macs-negative selection kit  (STEMCELL Technologies Inc)

Journal: The Journal of Clinical Investigation

Article Title: Delayed reinforcement of costimulation improves the efficacy of mRNA vaccines in mice

doi: 10.1172/JCI183973

Figure Lengend Snippet: ( A ) Experimental outline for evaluating whether treatment with α4-1BB on day 4 improves immune responses. Mice were immunized with 3 μg of an mRNA-spike vaccine followed by treatment with 50 μg of α4-1BB or control antibodies on day 4. ( B ) Summary of virus-specific CD8 + T cells. ( C ) Representative FACS plots of virus-specific CD8 + T cells. Data are from PBMCs. K b VL8 (shown in the y axis) is an MHC I tetramer used to detect SARS-CoV-2 spike–specific CD8 + T cells. Data are from 1 experiment, n = 4–5 per group/experiment; experiment was performed twice with similar results. Indicated P value in B was calculated with the Mann-Whitney test at the last time point.

Article Snippet: CD8 + T cells from Thy1.1 + P14 mice (PBMCs) were enriched using a CD8 MACS-negative selection kit (STEMCELL Technologies).

Techniques: Control, Virus, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Delayed reinforcement of costimulation improves the efficacy of mRNA vaccines in mice

doi: 10.1172/JCI183973

Figure Lengend Snippet: Experimental outline was similar to that in A. On day 7 after vaccination, splenic CD8 + T cells were MACS sorted. Subsequently, live CD8 + CD44 + K b VL8 tetramer + cells were FACS-purified to approximately 99% purity and used for bulk RNA-seq. ( A ) PCA shows transcriptional clustering. ( B ) Heatmap showing row-standardized expression of selected proliferation and apoptotic genes. ( C ) Heatmap showing row-standardized expression of selected cell cycle (top) and kinesins (bottom) genes. ( D ) Heatmap showing row-standardized expression of selected activation genes. ( E ) Heatmap showing row-standardized expression of selected effector genes. ( F ) GSEA plot showing enrichment of effector genes. ( G ) Validation of gene expression results at the protein level. Representative FACS plots showing the frequencies of virus-specific CD8 + T cells (K b VL8 + ) that differentiate into effector, effector memory, and central memory T cell subsets. ( H ) Pie diagrams showing CD8 + T cell subsets. ( I – K ) Numbers of central memory, effector memory, and effector CD8 + T cells. All data are from tetramer + (K b VL8 + ) cells from spleen. RNA-seq data are from 1 experiment, with n = 4 per group. Data in panel H are from 1 representative experiment, with n = 4 per group; the experiment was performed twice with similar results. All other data are from 2 experiments, with n = 4–5 per group/experiment. Indicated P values in I – K were calculated by the Mann-Whitney test.

Article Snippet: CD8 + T cells from Thy1.1 + P14 mice (PBMCs) were enriched using a CD8 MACS-negative selection kit (STEMCELL Technologies).

Techniques: Purification, RNA Sequencing, Expressing, Activation Assay, Biomarker Discovery, Gene Expression, Virus, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Delayed reinforcement of costimulation improves the efficacy of mRNA vaccines in mice

doi: 10.1172/JCI183973

Figure Lengend Snippet: Mice were immunized with 3 μg of each respective mRNA vaccine followed by treatment with 50 μg of α4-1BB or control antibodies on day 4. ( A ) Summary of LCMV-specific CD8 + T cell responses. ( B ) Representative FACS plots of LCMV-specific CD8 + T cells. ( C ) Pie diagrams showing CD8 + T cell subsets (gated on LCMV-specific CD8 + T cells). ( D ) Summary of OC43 spike–specific CD8 + T cell responses. ( E ) Summary of HIV env–specific CD8 + T cell responses. ( F ) Summary of OVA-specific CD8 + T cell responses. Data from A – C and F are after tetramer staining; data from D and E are after intracellular cytokine stimulation using overlapping peptide pools (IFN-γ + ). Data from A – F are from day 14 after vaccination, and are from 2 experiments, one with n = 5 per group/experiment and one with n = 2–5 per group/experiment. ( G ) Experimental outline for measuring 4-1BB following mRNA vaccination. P14 cells were transferred into C57BL/6 mice. One day after transfer, recipient mice were immunized with 3 μg of an mRNA-LCMV GP vaccine, and 4-1BB was measured on P14 cells at various time points. ( H ) 4-1BB on P14 cells after mRNA vaccination. Representative histograms showing 4-1BB expression on P14 cells. We utilized this P14 chimera model using a high number of P14 cells to allow us to detect 4-1BB expression on virus-specific CD8 + T cells at hyperacute points; endogenous virus-specific CD8 + T cells cannot be detected at hyperacute time points due to their low precursor frequency. Mean fluorescence intensity (MFI) is indicated on the x axis to denote “per-cell expression” of 4-1BB. This adoptive transfer experiment was performed 2 times, with n = 3 per group, showing similar results (peak of 4-1BB expression on day 4 after vaccination). All data are shown. Indicated P values in A and D – F were calculated by the Mann-Whitney test.

Article Snippet: CD8 + T cells from Thy1.1 + P14 mice (PBMCs) were enriched using a CD8 MACS-negative selection kit (STEMCELL Technologies).

Techniques: Control, Staining, Expressing, Virus, Fluorescence, Adoptive Transfer Assay, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Delayed reinforcement of costimulation improves the efficacy of mRNA vaccines in mice

doi: 10.1172/JCI183973

Figure Lengend Snippet: ( A ) Experimental outline to examine whether treatment with α4-1BB on day 4 improves immune protection by a therapeutic cancer vaccine. Mice were challenged s.c. with 2 × 10 6 B16-OVA tumor cells. On day 10 after tumor challenge, mice were vaccinated intramuscularly with 3 μg of mRNA-OVA. Mice received either control antibodies or α4-1BB (50 μg on day 0 or day 4 after mRNA-OVA vaccination). ( B ) Tumor control. ( C ) Survival. ( D ) Representative FACS plots showing CD8 + T cell responses on day 9 after vaccination. ( E ) Summary of OVA-specific CD8 + T cell responses on day 9. ( F – H ) Central memory, effector memory, and effector CD8 + T cells (K b SIINFEKL + PBMCs) at 2 weeks after vaccination. Data are from 2 experiments, one with n = 6–7 per group and one with n = 8 per group. Indicated P value in C was calculated by the log-rank (Mantel-Cox) test; all other P values were calculated by 2-way ANOVA with the Holm-Šídák multiple-comparison test.

Article Snippet: CD8 + T cells from Thy1.1 + P14 mice (PBMCs) were enriched using a CD8 MACS-negative selection kit (STEMCELL Technologies).

Techniques: Control, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Delayed reinforcement of costimulation improves the efficacy of mRNA vaccines in mice

doi: 10.1172/JCI183973

Figure Lengend Snippet: ( A ) Experimental outline for evaluating OX40 expression following mRNA vaccination. We utilized the same adoptive transfer model from G. ( B ) Kinetics of OX40 on virus-specific CD8 + T cells after mRNA vaccination. This adoptive transfer experiment was performed 2 times, with n = 3 per group, showing similar results (peak of OX40 expression on day 4 after vaccination). ( C ) Time-dependent effects of OX40 costimulation following mRNA vaccination. Mice were immunized with 3 μg of mRNA-spike vaccine, followed by treatment with OX40 costimulatory antibodies (200 μg of αOX40, clone OX-86) on day 0 or day 4 after vaccination. CD8 + T cell responses ( D ) and antibody responses ( E ) on day 15 after vaccination are shown. Data in D and E are from 3 experiments, with n = 5 per group. Indicated P values in D and E were calculated by Kruskal-Wallis test with Dunn’s multiple-comparison test.

Article Snippet: CD8 + T cells from Thy1.1 + P14 mice (PBMCs) were enriched using a CD8 MACS-negative selection kit (STEMCELL Technologies).

Techniques: Expressing, Adoptive Transfer Assay, Virus, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Delayed reinforcement of costimulation improves the efficacy of mRNA vaccines in mice

doi: 10.1172/JCI183973

Figure Lengend Snippet: Experimental outline was similar to that in A. On day 7 after vaccination, splenic CD8 + T cells were MACS sorted. Subsequently, live CD8 + CD44 + K b VL8 tetramer + cells were FACS-purified to approximately 99% purity and used for bulk RNA-seq. ( A ) PCA shows transcriptional clustering. ( B ) Heatmap showing row-standardized expression of selected proliferation and apoptotic genes. ( C ) Heatmap showing row-standardized expression of selected cell cycle (top) and kinesins (bottom) genes. ( D ) Heatmap showing row-standardized expression of selected activation genes. ( E ) Heatmap showing row-standardized expression of selected effector genes. ( F ) GSEA plot showing enrichment of effector genes. ( G ) Validation of gene expression results at the protein level. Representative FACS plots showing the frequencies of virus-specific CD8 + T cells (K b VL8 + ) that differentiate into effector, effector memory, and central memory T cell subsets. ( H ) Pie diagrams showing CD8 + T cell subsets. ( I – K ) Numbers of central memory, effector memory, and effector CD8 + T cells. All data are from tetramer + (K b VL8 + ) cells from spleen. RNA-seq data are from 1 experiment, with n = 4 per group. Data in panel H are from 1 representative experiment, with n = 4 per group; the experiment was performed twice with similar results. All other data are from 2 experiments, with n = 4–5 per group/experiment. Indicated P values in I – K were calculated by the Mann-Whitney test.

Article Snippet: On day 7, splenic CD8 + T cells were MACS-sorted with a MACS negative selection kit (STEMCELL Technologies).

Techniques: Purification, RNA Sequencing, Expressing, Activation Assay, Biomarker Discovery, Gene Expression, Virus, MANN-WHITNEY