Journal: Clinical Cancer Research

Article Title: Integrating Multisector Molecular Characterization into Personalized Peptide Vaccine Design for Patients with Newly Diagnosed Glioblastoma

doi: 10.1158/1078-0432.CCR-23-3077

Figure Lengend Snippet: T-cell characterization. A–C, immunoSEQ Analyzer differential abundance analysis displayed in pairwise scatter plots are shown for subjects 1, 2, and 3, respectively. The frequency of productive TCRβ rearrangements (clones) from the peripheral blood before and after NeoVax is compared. No data are available for subject 4 because they did not receive NeoVax. D–M, Bar graphs displaying IFNγ spots from IFNγ ELISPOT assays. Experiments were performed with duplicate or triplicate wells, and biological replicates were performed at least twice. *, P < 0.05 (Student t test) between pre- and post-NeoVax responses. There were 84, 77, and 35 days between pre- and post-NeoVax peripheral blood collections for subjects 1, 2, and 3, respectively. D, Direct ex vivo stimulation on select CD8 + T cells with minimal epitope peptide pools (m) or DMSO control for subject 1 post-NeoVax. E, Direct ex vivo stimulation on select CD4 + T cells with SLPs or DMSO control for subject 1 post-NeoVax. F, Direct ex vivo stimulation on select CD8 + T cells with minimal epitope peptide pools (m) or DMSO control for subject 1 pre-NeoVax. G, Direct ex vivo stimulation on select CD4 + T cells with SLPs or DMSO control for subject 1 pre-NeoVax. H–M, In vitro expanded PBMCs from pre- and postvaccination time points stimulated with IL2 and corresponding SLPs (green bars), minimal epitope peptide pools (dark orange bars), or DMSO control (purple bars) for 12 days prior to restimulation and analysis for subjects 1, 2, and 3.

Article Snippet: On day 13, cells were restimulated with 1 × 10 5 autologous T-cell depleted PBMCs (STEMCELL Technologies) and respective neoantigen minimal epitope peptide pools, the full-length peptide, or DMSO in a human IFNγ ELISPOT plate (ImmunoSpot).

Techniques: Clone Assay, Enzyme-linked Immunospot, Ex Vivo, Control, In Vitro

Journal: The EMBO Journal

Article Title: Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV ‐1 pathogenesis

doi: 10.15252/embj.2023114153

Figure Lengend Snippet: A CD127 + and CD127 − cells from Lin − CD56 − population after gating on lymphoid, singlet, live, CD45 + cells of PBMCs. Lineage (Lin) markers include antibodies against: CD3, CD4, TCRαβ, TCRγδ, CD19, CD20, CD22, CD34, FcεRIα, CD11c, CD303, CD123, CD1a, and CD14. B Heatmap of differentially expressed genes by RNA‐Seq, sorted Lin − CD56 − CD16 − CD127 + versus Lin − CD56 − CD16 − CD127 − cells, from PBMCs of five donors (log2 fold change > 1, P adj < 0.01 determined by DESeq2, the postsort profiles are shown in Appendix Fig ). C Normalized counts of Lin − CD56 − CD127 + (red) and Lin − CD56 − CD127 − (blue) cell‐related genes from (B) by DESeq2 ( n = 5). D, E Reactome analysis based on enriched transcripts of Lin − CD56 − CD127 + cells (D) or Lin − CD56 − CD127 − cells (E). F Gating of Lin − CD56 hi NK, Lin − CD56 dim NK, Lin − CD56 − NK cells, and Lin − CD56 − CD16 − CD127 + ILCs. G The indicated populations in (F) were detected with isotype controls or antibodies against TBX21, CRTH2, and RORγT. Data information: All data were generated using blood from healthy HIV‐1‐negative donors.

Article Snippet: UCB was processed as previously described and underwent CD3 T‐cell depletion (Brehm et al , ).

Techniques: RNA Sequencing, Generated

Journal: The EMBO Journal

Article Title: Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV ‐1 pathogenesis

doi: 10.15252/embj.2023114153

Figure Lengend Snippet:

Article Snippet: UCB was processed as previously described and underwent CD3 T‐cell depletion (Brehm et al , ).

Techniques: Control, Sequencing, Cell Stimulation, Multiplex Assay, Purification, Cell Isolation, Software

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil Cathepsin G Regulates Dendritic Cell Production of IL-12 During Development of CD4 T Cell Responses to Antigens in the Skin

doi: 10.4049/jimmunol.1800841

Figure Lengend Snippet: (A-C) Groups of 4 WT BALB/c and CXCR2−/− mice were sensitized with 0.25% DNFB on days 0 and +1. The indicated groups were treated with 200 μg anti-CD4, anti-CD8, or control rat IgG mAb on days −3, −2, and −1 prior to DNFB sensitization. On day +5, these mice and groups of nonsensitized mice were challenged on the ears with 0.2% DNFB. The change in ear thickness was determined (A) 24 hours later and every 24 hours thereafter for the sensitized and unsensitized (B) WT BALB/c or (C) CXCR2−/− mice and is shown as the mean increase in ear thickness for each group of 4 animals ± SEM. At 24 hours post-challenge: * P <0.05 and **P < 0.005 vs. untreated sensitized WT response; ***P <0.002 vs. untreated sensitized CXCR2−/− response. (D) On day +5 post-sensitization, lymph node (LN) cell suspensions were prepared from sensitized BALB/c or CXCR2−/− mice treated with 200 μg anti-CD4 or anti-CD8 mAb on days −3, −2, and −1 prior to DNFB sensitization. Aliquots of 5 × 105 of the LN cells were cultured with 5 × 105 DNBS-labeled syngeneic splenocytes to enumerate hapten-reactive cells producing IFN-γ or IL-17 by ELISPOT assay. The mean number of hapten-reactive T cells producing IFN-γ (black bars) or IL-17 (white bars) per 5 × 105 cells ± SEM for groups of 3 mice is shown. Results are representative of two individual experiments. (E) LN cell suspensions were prepared from sensitized WT BALB/c and CXCR2−/− mice and CD4 or CD8 T cells were removed from the total LN cell populations using anti-CD4 antibody coated or anti-CD8 antibody coated magnetic beads. Aliquots of 5 × 105 of the enriched CD4 or CD8 T cells were cultured with 5 × 105 DNBS-labeled syngeneic splenocytes and numbers of hapten-reactive cells producing IFN-γ- or IL-17-were determined by ELISPOT assay as above. Results are representative of two individual experiments.

Article Snippet: Antibodies for depletion of CD4 and CD8 T cells and Gr-1 + cells were purchased from BioXCell (West Lebanon, NH).

Techniques: Control, Cell Culture, Labeling, Enzyme-linked Immunospot, Magnetic Beads

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil Cathepsin G Regulates Dendritic Cell Production of IL-12 During Development of CD4 T Cell Responses to Antigens in the Skin

doi: 10.4049/jimmunol.1800841

Figure Lengend Snippet: (A-B) BALB/c mice were sensitized with 0.25 % DNFB on days 0 and +1. The indicated groups of mice were treated with 250 μg anti-Gr-1 mAb or anti-Ly6G mAb on days −1 and +1 during sensitization. On day +5 lymph node (LN) cell suspensions were prepared from the sensitized mice as well as nonsensitized (naïve) control mice. CD4 or CD8 T cells were removed from the total LN cell populations using anti-CD4 antibody coated or anti-CD8 antibody coated magnetic beads. Aliquots of 5 × 105 of the enriched CD4 or CD8 T cells were cultured with 5 × 105 DNBS-labeled syngeneic splenocytes to enumerate hapten-reactive cells producing IFN-γ or IL-17 by ELISPOT assay. The mean number of hapten-reactive CD4 (black bars) or CD8 T cells (white bars) producing IFN-γ or IL-17 per 5 × 105 cells ± SEM for groups of 4 mice is shown. (C-D) Groups of BALB/c mice were treated with 200 μg anti-CD8 mAb on days −3, −2, and −1 prior to sensitization with 0.25% DNFB and as indicated with 250 μg anti-Gr-1 or anti-Ly6G mAb on days −1 and +1 during sensitization. These mice and groups of unsensitized mice were challenged on the ears with 0.2% DNFB. The change in ear thickness was determined at 24-hour intervals post-challenge and is shown as the mean increase in ear thickness for each group of 4 animals ± SEM. *P < 0.001 when compared to responses in other sensitized groups.

Article Snippet: Antibodies for depletion of CD4 and CD8 T cells and Gr-1 + cells were purchased from BioXCell (West Lebanon, NH).

Techniques: Control, Magnetic Beads, Cell Culture, Labeling, Enzyme-linked Immunospot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil Cathepsin G Regulates Dendritic Cell Production of IL-12 During Development of CD4 T Cell Responses to Antigens in the Skin

doi: 10.4049/jimmunol.1800841

Figure Lengend Snippet: (A) Groups of WT C57/BL6 (black bar) or B6.IL-12−/− (white bar) mice were sensitized with 0.25 % DNFB on days 0 and +1. The indicated groups were treated with 200 μg anti-CD8 mAb on days −3, −2, and −1 prior to sensitization and as indicated with 250 μg anti-Gr-1 mAb on days −1 and +1. On day +5, all mice plus a group of nonsensitized mice were challenged on the ears with 0.2% DNFB. The change in ear thickness was determined 24 hours later and is shown as the mean increase in ear thickness for each group of 4 animals ± SEM. *P < 0.005 vs. response in untreated sensitized WT mice and **P not significant vs. response in untreated sensitized WT mice; ***P < 0.005 vs. response in untreated sensitized IL-12−/− mice. (B) Groups of WT C57/BL6 mice were sensitized with 0.25 % DNFB on days 0 and +1. The indicated groups were treated with 200 μg anti-CD8 mAb on days −3, −2, and −1 prior to sensitization and as indicated with 250 μg anti-Gr-1 mAb on days −1 and +1. The indicated groups were treated with nothing (black bars) or 250 μg anti-IL-12 mAb (white bars) on days 0, +1, +2, and +3. On day +5, these mice plus a group of nonsensitized mice were challenged on the ears with 0.2% DNFB and the change in ear thickness was determined 24 hours later as above. *P < 0.001 vs. response in untreated sensitized WT mice; **P < 0.001 vs. response in sensitized mice treated with anti-IL-12 mAb. (C) Groups of WT C57BL/6 mice were treated with 200 μg anti-CD8 mAb on days −3, −2, and −1 prior to sensitization with 0.25% DNFB and as indicated with 250 μg anti-Gr-1 or anti-Ly6G mAb on days −1 and +1. The indicated groups were also treated with 250 μg anti-IL-12 mAb or control rat IgG on days 0, +1, +2, and +3. On day +5 lymph node (LN) cell suspensions were prepared and aliquots of 5 × 105 cells were cultured with 5 × 105 DNBS-labeled syngeneic splenocytes to enumerate hapten-reactive cells producing IFN-γ or IL-17 by ELISPOT assay. The mean number of hapten-reactive T cells producing IFN-γ or IL-17 per 5 × 105 cells ± SEM for groups of 3 mice is shown for cells from mice treated with IgG (black bars) vs. anti-IL-12 mAb (white bars). Results are representative of two individual experiments. (D) WT C57/BL6 mice were sensitized with 0.25 % DNFB on days 0 and +1. The indicated groups of mice were treated with nothing (white bars) or with 250 μg anti-Gr-1 mAb (black bars) on days −1 and +1. On day +2, CD11c+ cells were purified from LN cell suspensions prepared from the sensitized mice and 3 × 105 CD11c+ cells were cultured with 5 × 105 LN cells from sensitized CD8-depleted mice. Where indicated 10 pg recombinant IL-12 was added to the cultures. After 72 hrs., supernatants were removed and tested by ELISA for production of IFN-γ. (E) Co-cultures of DC and enriched CD4 T cells isolated from LN of DNFB sensitized mice were established as in D above and after 72 hrs. supernatants were removed and tested by ELISA for production of IL-4, IFN-γ and IL-12. Where indicated 2 μg of anti-IL-12 mAb or control IgG was added to the culture.

Article Snippet: Antibodies for depletion of CD4 and CD8 T cells and Gr-1 + cells were purchased from BioXCell (West Lebanon, NH).

Techniques: Control, Cell Culture, Labeling, Enzyme-linked Immunospot, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil Cathepsin G Regulates Dendritic Cell Production of IL-12 During Development of CD4 T Cell Responses to Antigens in the Skin

doi: 10.4049/jimmunol.1800841

Figure Lengend Snippet: (A) Groups of 4 WT C57/BL6 mice or B6.CG−/− mice were sensitized with 0.25% DNFB and challenged on the ears with 0.2% DNFB on day +5 post-sensitization. The change in ear thickness was determined at 24-hour intervals post-challenge and is shown as the mean increase in ear thickness for each group ± SEM. (B) Groups of 4 WT C57/BL6 mice or B6.CG−/− were sensitized with 0.25 % DNFB on days 0 and +1. The indicated groups of mice were treated with 200 μg anti-CD8 mAb on days −3, −2, and −1 prior to sensitization. The sensitized mice and a group of nonsensitized mice were challenged with hapten on day +5 and the change in ear thickness determined 24 and 48 hours later and reported as in A. *P < 0.005 vs. response in untreated sensitized WT mice; **P < 0.05 vs. response in untreated sensitized CG−/− mice. (C) Groups of 4 WT C57/BL6 mice or B6.CG−/− mice were sensitized with 0.25% DNFB on days 0 and +1. The indicated groups were treated with 200 μg anti-CD4 or anti-CD8 mAb on days −3, −2, and −1 prior to DNFB sensitization. The sensitized mice and a group of nonsensitized mice were challenged with hapten on day +5 and the change in ear thickness determined 24 and 48 hours later and reported as in A. *P not significant vs. response in untreated sensitized WT mice and **P < 0.005 vs. response in untreated sensitized WT mice; ***P < 0.05 vs. response in untreated sensitized CG−/− mice. (E) CD11c+ DC were purified from the pooled LN of DNFB sensitized WT or CG−/− mice on day +2 after sensitization and aliquots of 5 × 105 DC were transferred i.d. to groups of naïve WT or CG−/− mice. Five days following DC transfer, mice injected with the DC and a group of nonsensitized naïve mice were challenged on the ears with DNFB and ear thickness was measured pre-challenge and at 24 hours after challenge and reported as in A. The indicated groups of mice receiving transferred DC were treated with anti-CD8 antibody or untreated. *P < 0.005 vs. responses in all other groups. Results are representative of two individual experiments.

Article Snippet: Antibodies for depletion of CD4 and CD8 T cells and Gr-1 + cells were purchased from BioXCell (West Lebanon, NH).

Techniques: Purification, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Neutrophil Cathepsin G Regulates Dendritic Cell Production of IL-12 During Development of CD4 T Cell Responses to Antigens in the Skin

doi: 10.4049/jimmunol.1800841

Figure Lengend Snippet: Groups of WT C57/BL6 mice and B6.CG−/− mice were sensitized with 0.25% DNFB on days 0 and +1. (A-B) Lymph node (LN) cell suspensions were prepared from these sensitized mice on (A) day +5 or (B) day + 7. On day +5 LN cell suspensions were prepared from the sensitized mice and CD4 or CD8 T cells were removed from the total LN cell populations using anti-CD4 antibody coated or anti-CD8 antibody coated magnetic beads. Aliquots of 5 × 105 of the enriched CD4 or CD8 T cells were cultured with 5 × 105 DNBS-labeled syngeneic splenocytes to enumerate hapten-reactive cells producing IFN-γ or IL-17 by ELISPOT assay from the sensitized WT (black bars) or CG−/− (white bars) mice. The mean number of hapten-reactive CD4 or CD8 T cells producing IFN-γ or IL-17 per 5 × 105 cells ± SEM for groups of 4 mice is shown. (C) Groups of WT C57/BL6 and B6.CG−/− mice were sensitized with 0.25 % DNFB on days 0 and +1. The indicated group of WT mice were treated with 250 μg anti-Gr-1 mAb on days −1 and +1 during sensitization. These sensitized mice were depleted of CD4 or CD8 cells by treatment with 200 μg depleting mAb on days −3, −2, and −1 prior to sensitization. On day +5 LN cell suspensions were prepared from the sensitized mice and CD4 or CD8 T cells were removed from the total LN cell populations using anti-CD4 antibody coated or anti-CD8 antibody coated magnetic beads and aliquots of 5 × 105 of the enriched CD4 (black bars) or CD8 (white bars) T cells were cultured with 5 × 105 DNBS-labeled syngeneic splenocytes. After 72 hours the culture supernatants were collected and the production of IFN-γ and IL-17 was assessed by ELISA. (D) Groups of WT C57/BL6 mice were sensitized with 0.25 % DNFB on days 0 and +1. The indicated groups of WT mice were treated with 250 μg anti-Gr-1 mAb on days −1 and +1 during sensitization and the indicated group of anti-Gr-1 mAb treated sensitized mice were given 200 ng active or boiled/inactive cathepsin G subcutaneously on days 0 and +1 during sensitization. On day +5 LN cell suspensions were prepared from the sensitized mice and CD8 T cells were removed from the total LN cell populations using anti-CD8 antibody coated magnetic beads. Aliquots of 5 × 105 of the enriched CD4 T cells were cultured with 5 × 105 DNBS-labeled syngeneic splenocytes to enumerate hapten-reactive cells producing IFN-γ or IL-17 by ELISPOT assay from the sensitized mice. The mean number of hapten-reactive CD4 T cells producing IFN-γ or IL-17 per 5 × 105 cells ± SEM for groups of 4 mice is shown.

Article Snippet: Antibodies for depletion of CD4 and CD8 T cells and Gr-1 + cells were purchased from BioXCell (West Lebanon, NH).

Techniques: Magnetic Beads, Cell Culture, Labeling, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay