Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: a Gating strategy in screening experiment using the panel of TCR Vβ antibodies. First, CD3 lymphocyte were separated into CD4 + and CD8 + populations using monoclonal antibodies (CD8 + : Pe-Cy7, CD4 + : PerCP). Next, the samples were stained with Vβ specific antibody cocktails included in IO Test Beta Mark TCR beta Repertoire Kit (Beckman-Coulter Immunotech, USA). The β variable chain family was determined based on FITC and PE positivity from CD4 + and CD8 + populations according to the manufacturer’s instruction. Vβ20 clone was detected from total CD4 + T cells (52.9%, middle panel) and total CD8 + T cells (1.74%, right). b Flow cytometry Vβ screening results from the index patient’s peripheral blood sample. T cell clonality with antibodies which target Vβ region of TCR was analysed of CD4 + T cells. The increased distribution suggests that the cells have large T cell clone. c Increased Vβ20 bearing clonotype over time in the index patient’s CD4 + T cells. Source data are provided as a Source data file. d T cell repertoire of FACS-sorted CD4 + Vβ20+ and CD8 + T cells analysed with TCRβ deep sequencing (Adaptive Biotechnologies). The TCRBV30-01 clone was detected in the CD4 + Vβ20+ fraction, but not in the CD8 + fraction. e Multicolor flow cytometry was applied to identify the immune phenotype of HSCT donor and index patient’s memory T cell subtypes. Central memory (CM), naïve, effector memory (EM), and terminal effector memory (TEMRA) cells. f The relative proportion of granzyme B positive (GrB + ) CD4 + T cells and GrB + CD8 + T cells in index patient. Index patient’s PBMCs were stained with anti-CD45, −CD3, −CD4, and −CD8 (surface markers), and then GrB stained after fixation and permeabilization. Stained cells were analyzed using FACSVerse.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Staining, Flow Cytometry, Sequencing

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Somatic mutations discovered in CD4 + T cells in the index patient, detected from 2013 sample.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: a Locations of mTOR, TLR2 , and NFκB2 somatic mutations. Linearized structure of MTOR, NFκB2 , and TLR2 presenting the location of somatic mutations. mTOR P2229R mutation is located in the kinase domain, NFκB2 P882Q in the C-terminus, and TLR2 W558L between LRR (Leucine-rich repeats) domain and transmembrane (TM) domain. b A heterozygous mTOR mutation (G to C, P2229R ) was detected in CD4 + T cells by Sanger sequencing. c Variant allele frequencies (VAFs) of mTOR P2229R , NFκB2 P882Q , and TLR2 W558L mutations in the index patient’s CD4 + T cells over time as measured with amplicon sequencing. Source data are provided as a Source data file. d VAFs (%) of mTOR P2229R mutation from the index patient’s skin, liver, and eyes biopsy. e Immunofluorescence staining indicated CD3 + CD4 + and CD3 + CD8 + T cell infiltration in the skin. Paraffin embedded skin biopsy from the index patient was sectioned and stained with antibody specific human CD3 (cyan), CD4 (green), and CD8 (red). White arrows indicate infiltrated CD3 + CD4 + or CD3 + CD8 + T cells. Original imaging magnification: 20×, the figure has been further zoomed to 25× for visualization. Scale bar: 50 μm.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Mutagenesis, Sequencing, Variant Assay, Amplification, Immunofluorescence, Staining, Imaging

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Somatic MTOR and NFκB2 mutations validated by amplicon sequencing in the index patient.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Amplification, Sequencing

Journal: Nature Communications

Article Title: Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease

doi: 10.1038/s41467-020-16115-w

Figure Lengend Snippet: Real-time cell analysing (RTCA) systems, xCELLigence TM , was applied to monitor real-time killing effect of primary fibroblasts obtained from the index patient and healthy donors. a Index patient’s primary fibroblasts were cultured as monolayers for 24 h to reach full confluence. Once confluent, the effector cells; NK-92 cell (positive control, 8:1), primary CD4 + T cells (4, 8, 16:1) and primary CD8 + T cells (16:1) with different ratios (effector cells:fibroblasts) were added to each well followed by co-culture (arrow indicates the point of adding the effector cells, 0 h). The control (black line, media) shows the impedance of the fibroblasts without any added effectors. The cell impedance was measured every 30 min for 21 h. The measured impedance was expressed as Cell Index with the normalization performed at time of addition of effector cells. b Visualization of monolayers of the primary fibroblast before and 21 h after addition of effector cells (8:1 for all effector cells). c CD4 + T cells and CD8 + T cells from healthy donors were added to healthy donors’ own fibroblasts ( n = 3). The cell impedance was measured for 21 h. Data is representative of three independent individuals. d Index patient’s fibroblasts were seeded with HLA1 and HLA2 antibodies (10 and 20 μg/mL). After CD4 + T cells (CD4 + T cells:fibroblasts = 8:1) were added the cell impedance was measured for 21 h. Dots represent mean values and error bars indicate range ( n = 2 for all conditions, technical duplicates). Source data are provided as a Source data file.

Article Snippet: Thereafter, HIER, peroxide and protein block were repeated, followed by application of anti-CD8 (1:500, Clone: C8/144B, Cat#: BSB 5174, Lot#: 5174JDL05, BioSB) primary antibody, HRP-conjugated secondary antibody diluted 1:3 with washing buffer and TSA 555 (PerkinElmer).

Techniques: Cell Culture, Positive Control, Co-Culture Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 1. OX40 expression on pDCs in the TME of HNSCC. (A) OX40 expression in the TME (measured by flow cytometry) of HNSCC patients on differ- ent immune cell subsets — pDCs (n = 89), cDCs (n = 53), CD8+ T cells (n = 16), CD4+ T cells (n = 17), CD4+ Th1 T cells (n = 12), and CD4+ Treg cells (n = 14). T cell subsets were gated from live CD45+CD3+ cells. Th1 cells were defined as CD4+Tbet+ T cells and Treg cells were defined as CD4+Foxp3+ cells. (B) Gating strategy for FACS analysis and sorting of OX40+ and OX40lo/– pDCs from patient specimens. After selecting for singlets and live cells, pDCs were gated from HLA-DRhiLineage– cells, followed by CD11c–CD123+ cells. pDCs were further confirmed by expression of CD303 (BDCA-2). OX40 expression on pDCs was determined using internal negative controls. (C) Immunofluorescence of pDCs in the TME demonstrating OX40 and CD123 coexpression. n = 4, with 4 patient repeats. Original magnification, ×63. Scale bar: 5 μm. Red, OX40; green, CD123; blue, DAPI. (D) OX40 expression on pDCs from different anatomic sites: PBMC (n = 17), dLN– (n = 50) or dLN+ (n = 59), and primary tumor (n = 53). (E) Correlation (Pearson, with a line of best fit) between OX40 and ICOSL expression on matched patient TME pDCs (n = 28). One-way ANOVA followed by Tukey’s post hoc test (A and D). **P < 0.01; ***P < 0.001; ****P < 0.0001. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (A, D, and E).

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 3. OX40+ pDCs promote antigen-specific CD8+ T cell responses. (A) Illustration of the antigen-specific in vitro coculture model, in which autologous OX40+/OX40lo/– pDCs from the TME/non-TME of HNSCC patients (n = 9) were cocultured with autologous TAA peptide-loaded mDCs and CD8+ T cells for 5 to 6 days, at which point antigen-specific CD8+ T cell responses were measured, (B) including for proliferation (eFluor 450–low) and IFN-γ production as demonstrated in flow plots of a patient’s CD8+ T cells cocultured with OX40+ or OX40lo/– pDCs sorted from their tumors. CD8+ T cell positivity was also measured for (C) Tbet and (D) eFluor 450–low in these coculture experiments. (E) CD8+ T cell positivity for CD69 after coculture with TAA peptide–loaded mDCs without pDCs (control) or with OX40+ or OX40lo/– pDCs from the TME versus non-TME (dLN–) (n = 5). (F) Illustration depicting the Transwell coculture assay in which OX40+ or OX40lo/– pDCs in the top chamber were separated from autologous CD8+ T cells and peptide-loaded mDCs in the bottom chamber. (G) Percentage of proliferating (eFluor 450–low) and GzB+ CD8+ T cells in Transwell versus contact coculture (n = 3). Representative flow plots show GzB production by CD8+ T cells cocultured with E7-loaded mDCs and OX40+ or OX40lo/– pDCs in coculture contact or separated by Transwell. (H) Flow plots comparing antigen presentation capacities of autologous OX40+ and OX40lo/– pDCs with mDCs, based on cytolytic CD8+ T cell responses (no peptide controls for these plots are shown in Supplemental Figure 2B). Shown is GzB production by CD8+ T cells in the presence or absence of OX40+/OX40lo/– pDCs (top) and IL-12p70 production by mDC/pDC subsets (bottom). n = 2; 2 experimen- tal repeats. One-way ANOVA followed by Tukey’s post hoc test (C–E and G). Bar graph data are mean ± SEM; *P < 0.05. NS, not significant. Middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points. Representative flow plots are shown (C–E and G).

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: In Vitro, Control, Co-culture Assay, Immunopeptidomics, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 4. The OX40-OX40L axis is utilized by pDCs in the TME. (A) OX40L expression measured by flow cytometry on OX40+ and OX40lo/– pDCs (n = 7). Correla- tion (Pearson, with line of best fit) of OX40 and OX40L expression intensities (per-cell normalized counts, total weighting) on pDCs measured on an HNSCC TME tissue section. n = 4; 4 patient repeats. (B) Immunofluorescence images from the TME showing a cell’s coexpression of OX40 (red) with CD123 (green), sitting adjacent to CD8-expressing (magenta) cells. Gallery view of Z-stacks (collected at 0.29-μm intervals). Original magnification, ×63. Scale bar: 5 μm. n = 4; 4 patient repeats. (C) Processed multispectral image (steps outlined in Supplemental Figure 2E for same example image) of the TME. Original magnification, ×40. Inset: a cluster CD123+ (green) cells, including one with OX40 expression (orange), next to a CD8+ (magenta) cell. (D) Representative image file written using Phenoptr to calculate touching pairs of phenotyped OX40L+CD68+ macrophages with phenotyped OX40+ pDCs. (E and F) Percentage of phenotyped OX40+/ OX40lo/– pDCs touching other OX40L+ cells, including pDCs, macrophages (Mφ), and “other cells” (Methods), in the TME and non-TME of patients (n = 3). (G) Ratios of CD8+ T cell counts within 30 μm of pDC subsets to total counts of CD123+ pDC subsets (OX40+/OX40lo/–) in the TME and non-TME of patients (n = 5). (H) Intercellular distances of phenotyped pDC subsets to the closest tumor margin (μm). Original magnification, ×40. n = 3; 3 patient repeats. One-way ANOVA followed by Tukey’s post hoc test (F) and unpaired (E), and paired (A, G, and H) t tests. *P < 0.05; **P < 0.01; NS, not significant. Bar graph data are mean ± SEM; middle line of box-and-whisker plot indicates the median, box limits indicate the first and third quartiles, and whiskers indicate “extreme” for all data points.

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Whisker Assay

Journal: Journal of Clinical Investigation

Article Title: OX40+ plasmacytoid dendritic cells in the tumor microenvironment promote antitumor immunity

doi: 10.1172/jci131992

Figure Lengend Snippet: Figure 6. OX40+ pDCs correlate to survival in cancer patients and suppress tumor growth. (A) Prospective recurrence-free survival (log-rank, Mantel-Cox test) of HNSCC cohort (n = 80), stratified by median (45%) intratumoral pDC OX40 expression, as measured by flow cytometry. (B) Overall survival (log-rank, Mantel-Cox test) of HNSCC patients (n = 500) from the GDC data portal, stratified first by median pDC gene signature Z scores followed by stratification of mean TNFRSF4 (encodes OX40) mRNA levels. (C) Correlation (Pearson, with line of best fit) of TNFRSF4 log2 mRNA levels (among cases with pDChi gene signatures) with CD8+ T effector scores in HNSCC (n = 172). (D) OX40 expression on intratumoral pDCs from different murine tumor mod- els. n =4; 4 experimental replicates. (E) gp100-specific Pmel-1 CD8+ T cell IFN-γ production by proliferating (eFluor 450–low) CD8+ T cells, measured in the presence or absence of pDCs from the dLNs of B16-F10– and B16CCR7-bearing mice. n = 2; 2 experimental repeats. (F) gp100-specific proliferating (eFluor 450–low) Pmel-1 CD8+ T cells in the presence or absence of B16CCR7 pDCs prestimulated with Resiquimod and OX86. n = 2; 2 experimental repeats. (G) Effect of pDC depletion (anti-PDCA1) in B16-F10– and B16CCR7-bearing mice compared with controls (anti-polyclonal IgG). Data are pooled from at least 2 independent experiments with 3 to 5 mice per group. (H) Quantification (by flow cytometry) of conventional cDCs (CD11c+CD11b–) and CD8a+ cDCs from B16CCR7-bearing mice treated with anti-PDCA1 or anti-polyclonal IgG. Data are pooled from individual experiments and normalized to 5 × 105 live cells. One-way ANOVA followed by Tukey’s post hoc test (D), 2-way ANOVA with Sidak’s test for multiple comparisons (H), and unpaired Student’s t test (G). **P < 0.01; ***P < 0.001. Tumor burden data and bar graph data are mean ± SEM.

Article Snippet: Tissue sections were incubated for 1 hour in blocking solution at 4°C with the following primary antibodies: rat anti–human CD8 (1:200, clone: YTC182.2, Bio-Rad), mouse anti–human CD123 (1:75, clone: 7G3, BD), and rabbit anti–human OX40 (1:80, E9U7O; Cell Signaling Technology).

Techniques: Expressing, Flow Cytometry