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 Experimental Medicine

Article Title: A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8 + T cell tolerance

doi: 10.1084/jem.20210759

Figure Lengend Snippet: STAT3 +/K392R drives expansion of the CD8 + effector compartment with up-regulation of cytokine and chemokine genes. (A) UMAP projection of reclustered T cells (CD3 + cells) from islet immune infiltrate used in scRNA-seq experiment (see ) with associated cell counts per cluster and comparison of cluster frequencies between the two genotypes (STAT3 +/K392R versus WT). γδT, γδ T cells. (B) Volcano plot showing STAT3 +/K392R versus WT differential gene expression in CD8 + clusters. FDR, false discovery rate. (C) Flow cytometry of Ccl5 protein expression in CD8 + memory T cells from the pLN in nondiabetic males at 14 wk ( n = 3 per group). (D) UMAP projection of reclustered CD8 + T cells. (E and F) Marker gene (E) or module score (F) expression in CD8 + T cell clusters. In F, genes used to calculate module scores for terminally exhausted T cells were Cd101 , Cd200r2 , Cd7 , Cd200r1 , and Il10 , and for transitory T cells, they were Cx3cr1 , Klrg1 , Il2ra , Il18rap , and S1pr5 , as previously described . (G) Relative population frequencies among nonnaive CD8 + cell clusters. (H) Volcano plot showing differential gene expression in CD8 + effector and transitory cell clusters relative to all CD8 + cells. Differential gene expression (B and H) based on nonparametric Wilcoxon rank-sum test. Volcano plots (B and H) use log N fold cutoff 0.25, and genes of interest are labeled. Data in C are representative of two independent experiments. Student’s t test (C) was used. Data are shown as mean ± SD. *, P ≤ 0.05.

Article Snippet: NOD cells were CD4 enriched using MACS EasySep CD4-negative selection kits (STEMCELL Technologies), and 8.3Tg + cells were naive CD8 enriched using MACS EasySep naive CD8-negative selection kits (STEMCELL Technologies).

Techniques: Expressing, Flow Cytometry, Marker, Labeling

Journal: The Journal of Experimental Medicine

Article Title: A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8 + T cell tolerance

doi: 10.1084/jem.20210759

Figure Lengend Snippet: scATAC-seq of infiltrating islet immune cells, additional characterizations of scTCR-seq data, and functional T cell adoptive transfer experiments. (A) UMAP projection of scATAC-seq profiles of T cells (CD3 + ) subclustered from CD45 + islet infiltrates of 8–10-wk-old male nondiabetic mice, WT ( n = 3, pooled), and STAT3 +/K392R ( n = 3, pooled). γδT, γδ T cells. (B) Normalized pseudobulk ATAC-seq tracks of CD8 + T cell clusters split by genotype around genes identified as up-regulated in corresponding scRNA-seq CD8 + T cell clusters. Peaks significantly up-regulated (from C) are shown in red. (C) Volcano plot showing differential peaks whose nearest gene is present in the significantly up-regulated genes between CD8 + T cell clusters (WT versus STAT3 +/K392R ). FDR, false discovery rate. (D) Volcano plot showing differential transcription factor motifs between CD8 + T cell clusters (WT versus STAT3 +/K392R ). (E) Transcription factor motif activity for CD8 + T cell clusters split by genotype. Motif sequence is shown above each violin plot. (F) TCR expression in UMAP clusters from subset of CD3 + T cells identified in scRNA-seq analysis. (G) Gini index showing clonal expansion in STAT3 +/K392R broken down by cluster and genotype. (H) Diabetes incidence after adoptive transfer of CD4 + BDC2.5 + Teff cells from mice without ( n = 6) and with STAT3 +/K392R ( n = 9) into NOD.Rag1 −/− mice (solid lines) and diabetes incidence after adoptive transfer of naive CD8 + T cells from WT 8.3Tg + mice with polyclonal CD4 + T cells from mice without ( n = 10) and with STAT3 +/K392R ( n = 5) into NOD.SCID mice (dotted lines). Results are pooled from two independent experiments for each set of incidence curves (solid lines and dotted lines), and a log-rank (Mantel-Cox) test was used.

Article Snippet: NOD cells were CD4 enriched using MACS EasySep CD4-negative selection kits (STEMCELL Technologies), and 8.3Tg + cells were naive CD8 enriched using MACS EasySep naive CD8-negative selection kits (STEMCELL Technologies).

Techniques: Functional Assay, Adoptive Transfer Assay, Activity Assay, Sequencing, Expressing

Journal: The Journal of Experimental Medicine

Article Title: A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8 + T cell tolerance

doi: 10.1084/jem.20210759

Figure Lengend Snippet: STAT3 +/K392R drives clonal expansion of diabetogenic CD8 + T cells. (A) Top 20 CD8 clones by count in each sample. (B) Top 10 specific CD8 clones with corresponding CDR3 sequences displayed. The CDR3 sequence for clone 6158 in red is nearly identical to that of the CD8-restricted TCR specific for islet-specific antigen IGRP (TCR-8.3; sequence shown below table). The only difference between clone 6158 and TCR-8.3 is a serine, denoted in blue, that replaces an alanine of the TCR-β chain. (C) Diabetes incidence of 8.3Tg + (IGRP-specific TCR) mice with ( n = 7) and without ( n = 11) STAT3 +/K392R confirms increased diabetogenicity. (D) Diabetes incidence after adoptive transfer of polyclonal WT CD4 + T cells with naive CD8 + T cells from 8.3Tg + mice with ( n = 5) and without STAT3 +/K392R ( n = 10) into NOD.SCID mice. Results are pooled from two independent experiments. A log-rank (Mantel-Cox) test (C and D) was used.

Article Snippet: NOD cells were CD4 enriched using MACS EasySep CD4-negative selection kits (STEMCELL Technologies), and 8.3Tg + cells were naive CD8 enriched using MACS EasySep naive CD8-negative selection kits (STEMCELL Technologies).

Techniques: Clone Assay, Sequencing, Adoptive Transfer Assay