Journal: The Journal of Biological Chemistry

Article Title: Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

doi: 10.1074/jbc.RA118.002649

Figure Lengend Snippet: C25-140 effect on proinflammatory signaling and T-cell activation. A, endogenous TRAF6 auto-ubiquitination (Ub) in MEF cells upon IL-1β stimulation is reduced after C25-140 treatment. B, C25-140 impairs IL-1β-induced IκBα phosphorylation. pIκBα levels were densitometrically quantified in relation to β-actin. Error bars, S.D.; n = 3 biological replicates were quantified; unpaired t test (two-tailed); ****, p < 0.0001. C, target gene (ICAM-1 and A20) expression is diminished after IL-1β stimulation and C25-140 treatment; error bars, S.D.; n ≥ 3 biological replicates; unpaired t test (two-tailed). *, p < 0.05; **, p < 0.01; ****, p < 0.0001. D, endogenous TRAF6 auto-ubiquitination after P/I stimulation is reduced after C25-140 treatment. E, C25-140 decreases IκBα phosphorylation after P/I stimulation in Jurkat T cells. pIκBα levels were densitometrically quantified in relation to β-actin. Error bars, S.D.; n = 3 biological replicates were quantified; unpaired t test (two-tailed). *, p < 0.05; **, p < 0.01. F, upon P/I stimulation, IL-2 and TNFα cytokine secretion is attenuated after C25-140 treatment. Error bars, S.D.; n = 3 biological replicates; unpaired t test (two-tailed). *, p < 0.05; **, p < 0.01; D, DMSO.

Article Snippet: Mouse studies Isolation of primary mouse CD4-positive T cells (ex vivo) CD4 + T cells from the peripheral lymph nodes of BALB/C mice were isolated by negative magnetic activation cell sorting selection using the CD4 + T-cell isolation kit II (Miltenyi).

Techniques: Activation Assay, Ubiquitin Proteomics, Phospho-proteomics, Two Tailed Test, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

doi: 10.1074/jbc.RA118.002649

Figure Lengend Snippet: C25-140 impedes immune receptor signaling in primary human PBMC and primary murine T cells. A–C, PBMCs from healthy individuals were isolated and treated with C25-140 before stimulation with IL-1β (A), LPS (B), and CD3/CD28 (C). Secretion of various NF-κB–driven cytokines was measured by ELISA and showed reduction of distinct cytokine secretion by C25-140 in all donors. D, primary mouse CD4+ T cells were isolated and treated with C25-140 and subsequently stimulated with CD3/CD28. IL-2 mRNA expression and protein secretion were measured using qRT-PCR and ELISA, respectively. C25-140 dose-dependently reduced IL-2 expression; error bars, S.D.; n = 4 biological replicates; signals were normalized to DMSO; unpaired t test (two-tailed). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; D, DMSO.

Article Snippet: Mouse studies Isolation of primary mouse CD4-positive T cells (ex vivo) CD4 + T cells from the peripheral lymph nodes of BALB/C mice were isolated by negative magnetic activation cell sorting selection using the CD4 + T-cell isolation kit II (Miltenyi).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Two Tailed Test

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: a , Illustration of TdT expression during lymphoid differentiation. b , Mass to charge ( m/z ) ratio spectra of TdT peptide-1 and -3. c , Intracellular staining of TdT in HLA-A2 pos moDCs after electroporation with mRNA encoding full-length TdT (red) or irrelevant control mRNA (black). HLA-A2 pos moDCs were then cocultured with HLA-A2 neg naïve CD8 + cells. d , Staining of CD8 + T cells with pMHC multimers complexed with peptide-1 or -3 (each multimer conjugated to both APC and PE, gating strategy shown in ) following cocultures with moDCs transfected with TdT or control mRNA. e , Staining of T-cell clones reactive to peptide-1 (clones 1–4) and peptide-3 (clone 1) showing the relevant pMHC multimers (green and purple) and corresponding nonrelevant pMHC multimers (black). f , Upregulation of CD137 on T-cell clones reactive to peptide-1 (green, clones 1–4) and peptide-3 (purple, clone 1) following coincubation with TdT neg HLA-A2 pos EBV-LCL cells pulsed with indicated concentrations of cognate peptides, or the B-ALL cell line NALM-6, naturally positive for TdT and HLA-A2. conc., concentration.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Expressing, Staining, Electroporation, Control, Transfection, Clone Assay, Concentration Assay

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: a , Histograms of viable CD8 + T cells transduced with T1 and T3 TCRs stained with anti-mouse TCR-β antibody or relevant peptide–MHC multimers. b , Activation of T1 and T3 cells following coincubation with peptide-pulsed T2 lymphoblast cells. Data are pooled from three independent experiments, with each circle representing the mean of three technical replicates in each experiment. Data are shown as mean ± s.d. c , CD137 upregulation on CD8 + T1 (green) and T3 (purple) cells after coculture with EBV-LCLs derived from one HLA-A2 pos and one HLA-A2 neg donor, either pulsed with relevant peptides or electroporated with mRNA encoding full-length TdT. d , Activation of CD8 + T1 and T3 cells after coculture with TdT pos cell lines REH (B-ALL) and HPB-ALL (T-ALL) in the presence/absence of introduced expression of HLA-A2, or pan-MHC class I blocking antibody W6/32 (Ab). e , IFN-γ production by T1 and T3 cells following coincubation with TdT pos NALM-6 cells (WT), or NALM-6 cells in which TdT 475-481 was deleted by CRISPR–Cas9 (KO) in the presence/absence of added peptide. f , Activation of T1 and T3 cells after coculture with various cell lines, with indicated HLA-A2 and TdT expression, loaded or not with TdT peptides (2 × 10 −7 M). The suffix + A2 denotes cell lines transduced with HLA-A*02:01. c - f , Results are from one experiment representative of two or three performed with different T-cell donors. Bars or connecting lines show mean, and individual data points represent either two ( e ) or three ( c , d , f ) technical replicates. g , Heat maps of IFN-γ produced by T1 (green) and T3 cells (purple) coincubated with EBV-LCLs pulsed with peptides from mimotope libraries. White circles, amino acid in wild-type peptide. IFN-γ concentration range for positive reactions was 500-31,254 pg ml -1 . One replicate per condition (see for correlation with CD137 activation assay). h , Model structures of TdT peptide-1 (green) and -3 (purple) represented as sticks, bound to the HLA-A2 molecule, in gray, shown from top (left) and side views (right). Individual amino acids are labeled with positional number and symbol.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Transduction, Staining, Activation Assay, Derivative Assay, Expressing, Blocking Assay, CRISPR, Produced, Concentration Assay, Labeling

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a) Gating strategy to analyze TCR transduced T cells (percentage of parental gate is shown). Top four panels: gating on FSC/SSC high , Live/Dead Fixable Near-IR neg , CD8 + singlets. Lower four panels: cells staining positively for APC and PE-labeled pMHC multimers complexed with either peptide-3 (left two plots: T3-transduced cells) or peptide-1 (right two plots: T1-transduced cells) and anti-mouse TCR-β antibody. Although >90% of T1 cells stain positively for the pMHC-PE multimer, a lower % was positive for the corresponding APC multimer due to lower fluorescence intensity of APC relative to PE. (b) Expansion of PB T cells after transduction with T1, T3 or 1G4 at indicated days after retroviral transduction. Data shown as mean ± s.e.m. of n = 3 (d3 and d7) or n = 2 (d5) HLA-A2 pos donors. (c) FACS plots showing phenotyping of T cells transduced with T1 on day 7 after spinoculation, gating on FSC/SSC high , Live/Dead Fixable Near-IR neg , CD3 + singlets, that are either CD4 + or CD8 + . Naïve, CM (central memory) and EM (effector memory) T cells. Bar graph in the lower right corner shows proportion of naïve, CM and EM CD4 + or CD8 + T cells on d7 after retroviral transduction with T1, T3 or control TCR. Inset numbers in the top right plot represent mean ± s.e.m. of all groups analyzed together due to similar proportions of CD8 + and CD4 + T cells in experimental groups. Data shown as mean ± s.e.m. of n = 3 donors. (d) Top: The region of gDNA in exon 10 which ends with the first 7 amino acids of the A2-presented epitope, selected as binding site for guide RNA. Middle: sequencing of gDNA from wild type NALM-6 cells, in which the TdT sequence corresponds to the sequence deposited in the database. Bottom: after cloning of CRISPR/Cas9-modified NALM-6 cultures, the selected clone shows a clean deletion in both alleles of the TdT gene, including the first 7 amino acids of the A2-presented epitope. (e) HLA-A2 expression on NALM-6 cells with TdT knock out (right) is unaffected relative to parental cell line (left).

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Staining, Labeling, Fluorescence, Transduction, Retroviral, Control, Binding Assay, Sequencing, Cloning, CRISPR, Modification, Expressing, Knock-Out

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a,b) Graphs depicting IFN-γ response of T1 (a) and T3 (b) cells to EBV-LCLs loaded with individual peptides from mimotope libraries containing a total of 161 9-mers for peptide-1 and 201 11-mers for peptide-3, at a concentration of (2 x 10 -7 M). Red dot in each graph represents response to wild-type peptide. Substituted amino acid in the original peptide is highlighted in red in the graph heading. IFN-γ concentration range for positive reactions was 500 – 31254 pg/mL (cut-off indicated by horizontal lines, one replicate per condition). (c) Correlation analysis between data for IFN-γ release, and percentage of cells expressing CD137, by T1 (left) and T3 cells (right) following activation by EBV-LCLs loaded with individual peptides from the mimotope libraries. r = Pearson correlation coefficient. (d) Peptide reactivity motifs for T1 and T3 that were queried in the ScanProsite search tool against human proteome databases. X indicates that any amino acid is allowed, while amino acids in square brackets [] indicate alternatives that are allowed for that given position in the peptide motifs. (e) Amino acid sequence of the human small integral membrane protein 19 (SMIM19), as listed in the non-curated TrEMBL database. (f) Percentage CD137 + events among T3 CD8 + T cells after 18 h co-culture with target cells loaded with indicated concentrations of either peptide-3 or SMIM19 peptide. Data are shown as mean of three technical replicates from one experiment representative of 2 performed.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Concentration Assay, Expressing, Activation Assay, Sequencing, Membrane, Co-Culture Assay

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: a , Viable TdT pos HLA-A2 pos NALM-6 and BV173 cells after 48 h of coculture with T1 and T3 cells (E/T ratio, 1/1), in percentage of corresponding numbers following treatment with mock-transduced T cells as quantified by flow cytometry. Data points represent technical replicates in one experiment representative of three performed. b , Experimental overview of BV173 and NALM-6 in vivo models. BLI ( c ) and quantification ( d ) of BV173-bearing mice 1 day before and 21 days after treatment with human T cells transduced with 1G4, T1 or T3, or left untreated. Survival analysis ( e ) and numbers ( f ) of TCR-transduced CD8 + T cells μl -1 blood at indicated time points in the aforementioned experimental groups. BLI ( g ) and quantification ( h ) of NALM-6-bearing mice 1 day before and 14 days after treatment with human T cells transduced with 1G4, T3, or left untreated. Survival analysis ( i ) and numbers ( j ) of TCR-transduced CD8 + T cells μl -1 blood at indicated time points in the aforementioned experimental groups. d , e , h , i , Data are pooled from two independent experiments: untreated, n = 10 (BV173) or n = 11 (NALM-6); 1G4, n = 7 (BV173) or n = 9 (NALM-6); T1, n = 8 (BV173); T3, n = 9 (BV173) or n = 11 (NALM-6). d , h , Box plots showing interquartile range (25th-75th percentile) with central bar indicating the median and whiskers indicating the range. Dots represent individual mice and data analyzed by one-way ANOVA with adjustment for multiple comparisons with Tukey’s post-test. e , i , Survival analysis performed by two-sided log-rank (Mantel-Cox) test. f , j , Data shown are from one representative experiment out of two performed. n = 5–6 mice per group. Connecting lines, mean; dots, individual mice.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Flow Cytometry, In Vivo, Transduction

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a) Flow cytometry histograms showing natural TdT or HLA-A2 expression in the B-ALL cell lines NALM-6 and BV173. (b) IFN-γ production in the co-culture supernatants of T1, T3 or mock T cells with indicated cell lines as measured by ELISA. (c) Expression of CD137 measured by flow cytometry by T1 and T3 cells after 18 h co-culture with indicated cell lines, loaded or not with relevant peptide and gated on CD8 + or CD4 + T cells. (d,e) Proliferation of PB T cells following transduction of T1, T3 or 1G4 in response to NALM-6 and BV173 following 5 days of co-culture at an E/T ratio of 1/1. Percentage proliferating cells is calculated based on events with low Cell Trace Violet staining out of total events that are FSC/SSC high , Live/Dead Fixable Near-IR neg , CD3 + singlets staining positively for either CD8 + or CD4 + . Bar graphs in (b), (c) and (e) show mean of three technical replicates from one experiment representative of 2 or 3 performed. (f) Gating strategy for the flow cytometry-based killing assay to enumerate BV173 tumor cells, here co-cultured with mock-transduced cells. Fluorescent beads (10,000) were added into each well and 5,000 beads were acquired for flow cytometry analysis. Live BV173 tumor cells were identified as Live/Dead Fixable Near-IR neg singlet cells that were CD3 - , CD8 - and CD19 + . (g) Flow cytometry plots of BV173 cells co-cultured with mock (black), T1 (green) and T3 cells (purple) for 48 h. Inset numbers display event counts within the live tumor cell gate.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Flow Cytometry, Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Transduction, Staining, Cell Culture

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a) Bioluminescence imaging (BLI) analysis of NSG mice on day 9 after BV173 ffluc-eGFP cell injection, one day prior to T-cell therapy. Data were pooled from two independent experiments, untreated n = 10, 1G4 n = 7, T1 n = 8, T3 n = 9. Data analysis by one-way ANOVA with Tukey’s multiple comparison test. (b,c) The percentage of human CD3 + (b) and CD8 + cells (c) in PB of mice analyzed at indicated time points out of total CD45 + human and CD45 + mice leukocytes. (d) Percent cells expressing T1, T3 and 1G4 TCRs among human CD8 + T cells throughout the experiment. Data in b-d are shown as mean ± s.e.m., n = 5/group. (e) Flow cytometry plots showing bone-marrow tumor burden in five T3-treated mice analysed on day 60 (M1-M5), compared to the 1G4-treated or untreated mouse at time of sacrifice (day 21). Threshold for positive leukemia detection was set as GFP + cells ≥ 0.01% of live viable cells. (f) Expression of HLA-A2 and TdT in leukemia cells harvested from the bone marrow of untreated, 1G4 and the one T3-treated mouse with detectable tumor burden (M5). Light gray histograms represent negative control. (g) Numbers of TCR-transduced CD8 + cells in blood of T3-treated mice on day 60 ( n = 5, M1-4: black circles, M5: red circle). Box plots in (a) and (g) show interquartile range (25 th to 75 th percentile) with central bar indicating the median and whiskers indicating the range while dots represent data from individual mice.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Imaging, Injection, Comparison, Expressing, Flow Cytometry, Negative Control

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a) BLI analysis of NSG mice on day 9 after NALM-6 ffluc-eGFP cell injection, one day prior to T-cell therapy. Data were pooled from two independent experiments, untreated n = 11, 1G4 n = 9, T3 n = 11. Data analysis by one-way ANOVA with Tukey’s multiple comparison test. (b,c) The percentage of human CD3 + (b) and CD8 + cells (c) in peripheral blood (PB) of mice analyzed at indicated time points out of total CD45 + human and CD45 + mouse leukocytes. (d) Percent cells expressing T3 and 1G4 TCRs among human CD8 + T cells throughout the experiment. Data in b-d are shown as mean ± s.e.m., n = 5 (1G4) and n = 6 (T3). (e) Flow cytometry plots showing bone-marrow tumor burden in one untreated and one 1G4-treated mice at time of sacrifice (day 14), and in five T3-treated mice sacrificed at end of experiment (day 57). (f) Expression of HLA-A2 and TdT in leukemia cells detected in bone marrow of untreated and 1G4-treated mice at the time of sacrifice. Light gray histograms represent negative control. (g) BLI images of mice in T3-treated group on day 57. Two of the T3-treated mice died for unknown reasons on day 35 and day 55 after T cell therapy, following negative BLI and PB flow cytometry analysis 5 days earlier, respectively, indicating that death was not related to leukemia. (h) IFN-γ levels in sera from untreated ( n = 5), 1G4 ( n = 4) or T3 ( n = 5) cell-treated mice on day 2 after T cell therapy, measured as mean fluorescence intensity (MFI) of IFN-γ capturing fluorescent beads. Statistical analysis by one way ANOVA with Tukey’s multiple comparison test. Box plots in (a) and (h) show interquartile range (25 th to 75 th percentile) with central bar indicating the median and whiskers indicating the range, while dots represent data from individual mice.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Injection, Comparison, Expressing, Flow Cytometry, Negative Control, Fluorescence

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a) TdT and HLA-A2 expression in diagnostic samples (PBMC) from three representative patients with B-ALL and T-ALL as analyzed by flow cytometry. Cytotoxicity data for these patients are shown in . (b) Percentage of CD137 + events among T1, T3 and mock-transduced CD8 + cells after 18 h culture with primary B-ALL and T-ALL patient samples that were either HLA-A2 pos TdT neg , HLA-A2 neg TdT pos or HLA-A2 pos TdT pos . Single positive patient samples were loaded or not with peptide-1 or -3 (concentration 10 -7 M) for 1 h before co-culture with corresponding T1 or T3 cells. FL: Follicular lymphoma, included as an HLA-A2 pos TdT neg control. Numbers below patient index show percentages of malignant blasts in the patient sample as analyzed by flow cytometry. Bar graphs in (b) show mean of 2-3 technical replicates run for individual patients.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Expressing, Diagnostic Assay, Flow Cytometry, Concentration Assay, Co-Culture Assay, Control

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: a , Representative t -SNE plots showing live HLA-A2 pos , TdT pos B-ALL tumor cells (CD19 + CD10 + events, left) and T-ALL tumor cells (CD5 + CD7 + CD99 + and surface CD3 − CD4 − events, right), normal B cells (CD19 + CD10 − ), normal T cells (CD3 + and CD8 + or CD4 + ) and CD34 + lin - progenitor cells following 72 h of coculture with mock-, T1- or T3-transduced T cells (E/T ratio, 1/1), as quantified by flow cytometry. b , Diagnostic samples from 12 patients (Pt.) with HLA-A2 pos , TdT B-ALL or T-ALL, assayed as described in a . Each dot represents the number of live tumor, normal B or T cells after coculture with T1 (green) or T3 (purple) cells, as percentage of corresponding numbers in cultures treated with mock-transduced T cells (gray). c , Dots showing numbers of live CD34 + lin − cells after coculture with T1 (green) and T3 (purple) cells as percentage of corresponding numbers in cocultures treated with mock-transduced T cells (gray). b,c, Data points represent three or four technical replicates and horizontal lines denote mean. Data shown are from one experiment representative of at least two performed for each patient sample. d, t -SNE plot of PB diagnostic sample from B-ALL patient no. 1N after 72 h of coculture with autologous T cells transduced with T1, T3 or mock. a,d, Inset numbers denote absolute event counts of the indicated cell populations. e, Tandem mass spectrometry fragmentation spectra of TdT peptide-1 and -3 identified from leukemia cells of patient no. 119N. f, Expression of CD19 and TdT in leukemia cells before and after relapse from CD19-specific CAR T-cell therapy in two patients with B-ALL. Ctrl, control. g, Expression of TdT in leukemia cells from a patient with T-ALL at the time of primary diagnosis (Dx) and at relapse after chemotherapy.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Flow Cytometry, Diagnostic Assay, Transduction, Mass Spectrometry, Expressing, Control, Biomarker Discovery

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a) Transduction efficiency of the T1 (green) and T3 (purple) TCRs in normal CD8 + T cells from an HLA-A2 pos TdT pos patient diagnosed with B-ALL. Histograms are gated on live, CD8 + T cells. (b) T1 and T3 cell activation after 18 h co-culture with the autologous blasts. (c) Quantification of malignant cells, normal B, T and CD34 + lin - progenitor cells after performing the flow cytometry-based cytotoxicity assay for 72 h. Data points represents technical replicates (3-4) from one representative experiment out of 2 performed and horizontal lines show mean. (d) Flow cytometry dot plots from one of the test replicates in c, showing gating strategy to identify tumor blasts (CD19 + CD10 + ), normal B cells (CD19 + CD10 - ), T cells (CD19 - CD10 - CD3 + ) and CD34 + progenitor cells (CD19 - CD10 - CD3 - CD34 + ). Populations were gated and overlaid on a t-SNE plot with designated colors as indicated (left view). Inset numbers in d show event count of tumor cells after co-culture with mock, T1 and T3 cells.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Transduction, Activation Assay, Co-Culture Assay, Flow Cytometry, Cytotoxicity Assay

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: a , Experimental overview of the PDX model. b , Percentage leukemic cells adjusted for human T cells in PB at baseline and at indicated time points after T-cell infusion. c , Representative FACS plots of viable single MNCs from BM of T3-treated (top) and DMF5-treated (bottom) NSG mice engrafted with primary human B-ALL cells. d , Percentage leukemic cells (hCD45 + CD19 + CD10 + ) adjusted for human T cells at baseline (BM) and at terminal analysis on day 11 after T-cell infusion (BM, PB and spleen). e , Number of leukemic cells present in BM. f , Number of TCR-transduced human CD8 + cells in BM, PB and spleen. g , Total numbers of human- and mouse-derived MNCs in BM, PB and spleen. h , Number of mouse CD45 + cells in BM. b , d - h , Data are pooled from two independent experiments and presented as mean ± s.e.m. of untreated ( n = 5), DMF5-treated ( n = 8) and T3-treated ( n = 8) mice. Populations identified by flow cytometry according to gating strategy shown in c . Kruskal-Wallis one-way ANOVA by Dunn’s multiple comparisons test ( d , e ) and two-tailed Mann-Whitney test ( f - h ) were performed for statistical analyses.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Derivative Assay, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: a , b , FACS analysis of viable human CD19 - HLA-DR neg thymocytes ( a ), and spaghetti-plot illustrating HLA-A2 and TdT mean fluorescence intensity (MFI) at distinct stages of T-cell development ( b ), defined as shown in , in human thymus. c , Experimental overview of humanized NSG (hu-NSG) mouse model used to investigate the effect of T3-cell therapy on healthy human hematopoiesis. d , Percentage of human TdT’” cells in thymus of hu-NSG mice ( n = 8 per group). e , HLA-A2 mean fluorescence intensity of TdT’” cells (hCD45 + TdT) and surface CD3 + (s-CD3 + ) human thymocytes derived from one normal human thymus and from thymus of 1G4 ( n = 8) and T3 ( n = 8)-treated humanized mice. f , Percentage of TCR-transduced CD8 + cells in PB at indicated days after T-cell infusion ( n = 8 per group). g , Myeloid and erythroid colonies generated from sorted normal adult human BM CD34 + cells ( n = 4 biological replicates, data pooled from two independent experiments) following coculture with or without 1G4, T1 or T3 cells for 72h at an E/T ratio of 2/1. All data are presented as mean±s.e.m.; dots in d - f represent individual mice at terminal analysis on day 17 post treatment, unless otherwise stated.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Fluorescence, Derivative Assay, Generated

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a) Flow cytometric analysis of thymocytes from normal human thymus removed from a four months old child with congenital cardiac defect (but otherwise healthy) in conjunction with cardiac surgery. Flow plots show gating strategy to define four key developmental stages during thymocyte differentiation, including 1) early double negative (DN), 2) late double negative, 3) double positive (DP) and 4) single positive (SP, either CD4 + or CD8 + ) thymocytes. Arrows within the FACS plots in third row indicate direction of differentiation with regard to expression of TdT and HLA-A2 during these developmental stages, illustrated with continuous lines in . (b) Percentage of TCR-transduced cells among CD8 + T cells isolated from spleens of three humanized NSG mice engrafted with healthy cord blood following transduction and expansion, prior to infusion into littermates day 0, as illustrated in . (c) Functionality of human TCR-transduced T cells shown in (b), as determined by the number of viable BV173 cells present after 72 h co-culture, in percent of corresponding numbers following treatment with 1G4 cells, quantified by flow cytometry (E/T ratio of 1/1). Co-cultures were started on the same day as T cells were injected into mice. Data points show 2 technical replicates from one experiment. (d,e) Representative FACS plots of viable single hCD45 + CD19 - CD33 - thymocytes from 1G4 or T3-treated humanized NSG mice at terminal analysis on day 17 post T cell infusion illustrating surface CD3 and TdT expression (d) and HLA-A2 and TdT expression (e). Inset numbers show mean ± s.e.m., n = 8 per group.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Expressing, Isolation, Transduction, Co-Culture Assay, Flow Cytometry, Injection

Journal: Nature biotechnology

Article Title: T cells targeted to TdT kill leukemic lymphoblasts while sparing normal lymphocytes

doi: 10.1038/s41587-021-01089-x

Figure Lengend Snippet: (a,b) Representative FACS plots from terminal analysis of viable single MNCs in PB, BM, spleen and thymus from humanized NSG mice treated with 1G4 (a) and T3 (b) cells, to determine the percentage of TCR-transduced T cells and lineage distribution (myeloid: CD33 + , B cells: CD19 + , T cells: CD3 + CD4 + or CD3 + CD8 + ). (c,d) Percentage human CD45 + cells (out of human and mouse CD45 + cells) in PB at indicated days after T cell infusion (c), and in BM, spleen and thymus of humanized NSG mice treated with 1G4 or T3 cells at terminal analysis on day 17 (d). (e,f) Percentage lineage distribution within human CD45 + engrafted cells in PB at indicated days after T cell infusion (e) and in BM, spleen and thymus at terminal analysis (f). (g) BM cellularity at terminal analysis. n = 8 per group in c-g. (h) Myeloid and erythroid colonies generated from sorted normal human BM CD34 + Lineage - cells from one donor co-cultured without T cells (control), or with 1G4, T1 or T3 cells for 48 h in the presence of peptide-1 or peptide-3 at an E/T ratio of 2/1 (2 technical replicates per group). Data are presented as mean ± s.e.m.

Article Snippet: On day -1, naïve CD8 + T cells were isolated from PBMCs from HLA-A2 neg donors by the AutoMACS Pro Separator and CD8 + T-cell isolation kit premixed with CD45RO- and CD57-reactive beads (Miltenyi Biotec).

Techniques: Generated, Cell Culture, Control

Journal: bioRxiv

Article Title: Combining Cell-Intrinsic and -Extrinsic Resistance to HIV-1 By Engineering Hematopoietic Stem Cells for CCR5 Knockout and B Cell Secretion of Therapeutic Antibodies

doi: 10.1101/2024.03.08.583956

Figure Lengend Snippet: a) Diagram for editing and engraftment of CB CD34 + HSPCs in NSG mice to characterize engraftment capacity and multilineage potential. b) Distribution frequency of WT, INDEL, and KI alleles in CB CD34 + HSPCs prior to transplantation in NSG mice. Cells were targeted at CCR5 with an AAV6 MOI of 1250 for each antibody construct (n = 1 pooled sample from 5 donors). c) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the 10-1074+Ibalizumab targeted cells shown in panel b (n = 1). d) Percent human cell chimerism in the bone marrow at 16 weeks post-transplantation (n = 5 for mock, 10-1074, and 10-1074+Ibalizumab, n = 4 for Ibalizumab). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test (ns, not significant, P = 0.3287; *P = 0.0496; **P = 0.0082). e) Percent human cell chimerism in the spleen at 16 weeks post-transplantation (n = 4 for mock, 10-1074, and 10-1074+Ibalizumab, n = 3 for Ibalizumab). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test (ns, not significant, P = 0.0806 for Mock vs 10-1074, P = 0.1363 for Mock vs Ibalizumab; *P = 0.0216). f) Percent of human cells in the bone marrow (n are the same as in d) or g) spleen (n are the same as in e) that are CD19 + (B cell lineage), CD33 + (myeloid cell lineage), or within other lineages in mice engrafted with mock (black) or gene edited HSPCs (10-1074, blue; Ibalizumab, purple; 10-1074 and Ibalizumab, red). h) Percent of human alleles from the bone marrow or spleen with knock-in of the indicated antibody constructs (n are the same as in d and e for bone marrow and spleen, respectively). i) Percent of human alleles with knock-in from the bulk bone marrow (as shown in panel h) or in positively selected bone marrow CD19 + cells (n are the same as in d). Lines connect dots representing measurements from the same mice. Two-tailed Mann-Whitney test (*P = 0.0135). j) Percent of human alleles from the bone marrow with an INDEL at CCR5 (n are the same as in d). This analysis does not include alleles with KI. All bars represent mean. All dots represent individual mice engrafted with mock HSPCs or HSPCs edited with AAV6 and RNP for the antibody construct(s) indicated.

Article Snippet: Humam CD19 + cells were isolated from the bone marrow using positive selection with the CD19 MicroBeads, human kit (Miltenyi Biotec, cat.: 130-050-301).

Techniques: Transplantation Assay, Construct, Knock-In, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Combining Cell-Intrinsic and -Extrinsic Resistance to HIV-1 By Engineering Hematopoietic Stem Cells for CCR5 Knockout and B Cell Secretion of Therapeutic Antibodies

doi: 10.1101/2024.03.08.583956

Figure Lengend Snippet: a) Distribution frequency of WT, INDEL, and KI alleles in CB CD34 + HSPCs prior to transplantation in NBSGW mice. Cells were targeted at CCR5 with an AAV6 MOI of 625 for each linker antibody construct and treated with 0.5 µM AZD7648 (n = 2 independent biologic donors; data also shown in ). b) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the 10-1074+Ibalizumab targeted cells shown in panel a (n = 2). c) Percent human cell chimerism in the bone marrow 12 weeks post-transplantation, Two-tailed Mann-Whitney test (**P = 0.0040, n = 4 for mock and n = 8 for 10-1074+Ibalizumab). d) Percent of human cells in the bone marrow that are CD19 + (B cell lineage), CD33 + (myeloid cell lineage), or within other lineages in mice engrafted with mock (black) or gene edited (red) HSPCs (n are the same as in c). One-way ANOVA Kruskal-Wallis test plus Dunn’s multiple comparisons test (ns, not significant, P = 0.1650 for CD33+ comparison, P = 0.3492 for Other comparison; *P = 0.0474). e) Percent of human alleles with knock-in from the bulk bone marrow or in positively selected bone marrow CD19 + cells. Lines connect dots representing measurements from the same mice (n are the same as in c). Two-tailed Mann-Whitney test (ns, not significant, P = 0.7178). f) Percent of human alleles from the bone marrow with an INDEL at CCR5 (n are the same as in c). This analysis does not include alleles with KI. All bars represent mean and all error bars represent SD. All dots represent individual mice engrafted with mock HSPCs or HSPCs edited with AAV6 and RNP.

Article Snippet: Humam CD19 + cells were isolated from the bone marrow using positive selection with the CD19 MicroBeads, human kit (Miltenyi Biotec, cat.: 130-050-301).

Techniques: Transplantation Assay, Construct, Two Tailed Test, MANN-WHITNEY, Comparison, Knock-In

Journal: bioRxiv

Article Title: Combining Cell-Intrinsic and -Extrinsic Resistance to HIV-1 By Engineering Hematopoietic Stem Cells for CCR5 Knockout and B Cell Secretion of Therapeutic Antibodies

doi: 10.1101/2024.03.08.583956

Figure Lengend Snippet: a) Allelic integration frequency in adult peripheral blood CD19 + B Cells targeted with AAV6 cassettes for linker antibodies as indicated. Each AAV6 was used at an MOI of 25,000 (dots represent independent biologic donors, n = 9 for mock and 10-1074, n = 5 for Ibalizumab and 10-1074+Ibalizumab, n = 3 for PGDM1400 and CAP256V2LS). b) Percent of KI alleles integrated with 10-1074 or Ibalizumab within the “10-1074+Ibalizumab” targeted cells shown in panel a (n = 5). c) Measured IC 50 for each antibody against TRO11 or CNE55 pseudoviruses as determined by TZM-bl assay in (NN, not-neutralizing). d) Inhibition of infection with TRO11 or e) CNE55 HIV-1 pseudovirus by culture supernatant from B cells engineered to express linker antibodies as indicated. Percent infection for each dose of supernatant from gene targeted B cells is normalized to infection at each dose of supernatant from mock B cells from the same donor (n = 3 biological donors, data points and error bars represent mean with standard deviation of technical duplicate infections).

Article Snippet: Humam CD19 + cells were isolated from the bone marrow using positive selection with the CD19 MicroBeads, human kit (Miltenyi Biotec, cat.: 130-050-301).

Techniques: Inhibition, Infection, Standard Deviation

Journal: Cells

Article Title: Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

doi: 10.3390/cells10082081

Figure Lengend Snippet: Purification and characterization of EV preparations from EwS and fibroblast cell lines and healthy donor plasma. ( A ) Size distribution of EVs isolated by ultrafiltration and differential centrifugation from healthy donor plasma, MRC5 fibroblasts, and A4573, A673, TC32, and TC71 EwS cell lines. Nanoparticle tracking analysis of three independent EV preparations (dotted line) and respective mean (solid line) is shown. ( B ) Immunoblotting-detection of chaperon proteins Hsp70 and Hsp90, EV markers CD63, CD81 and TSG101, tubulin and actin in the purified EV preparations from EwS cell lines and healthy donor plasma ( n = 3 independent donors). ( C ) Expression of the CD63 and CD81 EV markers (solid line) compared to IT antibodies (dotted line) on MRC5 EVs or EwS EVs bound to 3.9 µm latex beads. Representative flow cytometry result (a total of 3 independent EV preparations) is shown. ( D ) TNF release from CD14 + monocytes treated for 6 h with three independent EV preparations (3 × 10 9 EVs/mL) from healthy donor plasma, MRC5, or EwS cell lines. Conditioned medium was analyzed by cytokine singleplex assay.

Article Snippet: CD14 + or CD33 + myeloid cells were isolated by positive selection using anti-human CD14 magnetic particles (BD Biosciences, Heidelberg, Germany) or anti-human CD33 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol.

Techniques: Purification, Clinical Proteomics, Isolation, Centrifugation, Western Blot, Expressing, Flow Cytometry, Singleplex Assay

Journal: Cells

Article Title: Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

doi: 10.3390/cells10082081

Figure Lengend Snippet: EwS EVs induce pro-inflammatory responses in myeloid cells. ( A – F ) Cytokine and chemokine multiplex profiling of the conditioned medium from myeloid cells treated with PBS control (Ctrl) or EVs from plasma of healthy donors, MRC5 fibroblasts, or EwS cell lines, as indicated. ( A ) CD33 + myeloid cells and ( B ) CD14 + monocytes were treated with 3 × 10 9 EVs/mL from A4573, A673, and TC32 EwS cell lines or PBS for 24 h. ( C ) CD33 + myeloid cells were incubated for 24 h with 5 × 10 8 to 7.5 × 10 9 EVs/mL from an A4573 cell line or healthy donor plasma. ( D ) CD33 + myeloid cells were incubated with 5 × 10 8 to 7.5 × 10 9 EVs/mL from A673 EwS cells or MRC5 fibroblasts. The medium was changed after 6-h treatment and analyzed 18 h later. ( E ) CD14 + myeloid cells were incubated with 3.75 or 7 × 10 9 EVs/mL from TC32 and TC71 EwS cells or PBS. The medium was changed after 6-h treatment and analyzed 18 h later. ( F ) CD33 + myeloid cells were differentiated with the IL-4 and GM-CSF cocktail in the presence of 3 × 10 9 EVs/mL from TC32 or MRC5 cells, or PBS. Conditioned medium was analyzed on days 3 and 5. Results were obtained from one ( E ), two ( C ), or three ( A , B , D , F ) independent donors with one ( D ), two ( C ), or three ( A , B , E , F ) independent EV preparations. Data are presented as mean ± SD. An unpaired two-tailed t -test was used to calculate p values ( E ). * p ≤ 0.05.

Article Snippet: CD14 + or CD33 + myeloid cells were isolated by positive selection using anti-human CD14 magnetic particles (BD Biosciences, Heidelberg, Germany) or anti-human CD33 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol.

Techniques: Multiplex Assay, Control, Clinical Proteomics, Incubation, Two Tailed Test

Journal: Cells

Article Title: Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function

doi: 10.3390/cells10082081

Figure Lengend Snippet: EwS EVs affect myeloid cells at an early stage of differentiation and impair their maturation. ( A ) Experimental design ( B – D ). CD14 + or CD33 + myeloid cells purified from healthy donors were differentiated in the presence of GM-CSF and IL-4 for 5 days and matured with the IL-1β, IL-6, PGE 2 , and TNF cocktail for additional 2 days to moDCs. The indicated EVs (3 × 10 9 /mL) or PBS control (Ctrl) were added at day 0 of differentiation. ( B – E ) Flow cytometry analysis of the indicated maturation markers at day 7. ( B ) Geometric mean fluorescence intensity (gMFI) of CD80, CD86, and HLA-DR normalized to IT of CD14 + monocytes treated with EVs from MRC5, A673, TC32, or TC71 cells, or PBS. Fold change to Ctrl is shown. ( C ) gMFI of CD80, CD86, and HLA-DR normalized to IT of CD14 + myeloid cells treated with three independent EV preparations from TC32 cells or PBS. ( D ) gMFI of CD80, CD86, and HLA-DR normalized to IT of CD33 + myeloid cells treated with MRC5 EVs, TC32 EVs, or PBS. ( E ) TC32 EVs were added to CD14 + or CD33 + myeloid cells during differentiation (day 0 and 3) or maturation (day 5). Representative pseudocolor plots of CD80, CD83, CD86, and HLA-DR compared to the respective IT antibodies are shown. The results were obtained from one ( C ) or three ( B , D , E ) independent donors with three ( B – E ) independent EV preparations. Data are presented as mean ± SD. One-way ANOVA with multiple comparison Turkey test ( B ), and unpaired ( C ) and paired ( D ) two-tailed t -tests were used to calculate p values. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.001.

Article Snippet: CD14 + or CD33 + myeloid cells were isolated by positive selection using anti-human CD14 magnetic particles (BD Biosciences, Heidelberg, Germany) or anti-human CD33 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol.

Techniques: Purification, Control, Flow Cytometry, Fluorescence, Comparison, Two Tailed Test

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: T-cell responses against Bax peptides and mapping the specificity of the CD8 + T-cell clone 6C5. (a) Purified CD8 + T cells were cultured with irradiated autologous activated B-CLL cells and Bax peptides 601–23 for 5 weeks before testing by interferon- γ (IFN- γ ) ELISpot. Antigen-presenting cells (APC) were autologous activation B-CLL cells. Numbers shown are spots/10 5 T cells (mean of triplicates ± SD, n = 1) Statistical analysis (unpaired two-tailed t -test) was carried out using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). (b) T-cell cultures generated by limited dilution were tested for the recognition of Bax peptides 601–23 by IFN- γ ELISpot. T cells were plated (∼2 × 10 4 to 3 × 10 4 /well) with APC or with APC + peptides at a 1 : 1 ratio. APC were T2 cells. Background responses (T cells + T2) were subtracted from the data ( n = 1). (c) 6C5 was tested by IFN- γ ELISpot against the Bax peptide pool 601–23, split pools, and individual peptides. T cells were plated (1 × 10 4 /well) in triplicate with T2 or T2 + peptides at a 1 : 1 ratio. Background response (T cells + T2) was subtracted from the data (5 SFC/10 4 cells) (mean ± SD of triplicates, n = 2). (d) 6C5 was assayed against T2 cells pulsed with varying concentrations (20–6·25 μg/ml) of Bax P603 and Bax P605 at 1 : 1 ratio for 18 hr. Cell-free supernatants were harvested and tested for the presence of IFN- γ by ELISA. The EC 50 value was calculated using the fitted curve, P603 – 4·92 µ m and P605 > 100 µ m (mean ± SD of duplicates, n = 3).

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Purification, Cell Culture, Irradiation, Enzyme-linked Immunospot, Activation Assay, Two Tailed Test, Software, Generated, Enzyme-linked Immunosorbent Assay

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: 6C5 recognition of crude P603 but not highly purified P603. Representative sample of flow cytometry analysis (three independent experiments) of intracellular staining of interferon- γ (IFN- γ ). T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + Bax peptide P603 (> 95% Pure, 10 μg/ml) or P603 (77% Pure, 10 μg/ml) at a 1 : 1 ratio for 5 hr. Lymphocytes were gated based on their forward and side scatter profile and then doublet exclusion was performed based on forward scatter height versus forward scatter width. T cells were then gated on CD3 + CD8 + cells and IFN- γ production was assessed through intracellular staining with anti-IFN- γ (mean ± SD of duplicates, n = 3). Statistical analysis (unpaired two-tailed t -test) was carried out using GraphPad Prism .

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Purification, Flow Cytometry, Staining, Cell Culture, Two Tailed Test

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: 6C5 recognizes a peptide present in fraction 8. (a) CD8 + T-cell clone 6C5 was cultured in the presences of various peptide preparations representative of potential C-terminal (GTPT) and N-terminal (LSYF) deletions, as well as C-terminal additions. T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + peptides at a 1 : 1 ratio for 5 hr. Interferon- γ (IFN- γ ) production was assessed through intracellular staining with anti-IFN- γ . To facilitate gating, the cultures were also co-stained with anti-CD3 and anti-CD8. Background (T-cells + T2) were subtracted from the data (mean ± SD, n = 3). (b) Fractionation was performed by HPLC (Waters 2525 pump and 2996 detector) with a Vydac 218TP 250 × 22 mm column. Peptides were eluted with a binary gradient of 0 min 95% A, 1 min 95% A, 16 min 50% A where solvent A = H 2 O, 0·05% trifluoroacetic acid (TFA) and solvent B = acetonitrile, 0·05% TFA at a flow rate of 22·9 ml/min. The chromatogram shows the absorbance at 220 nm. (c) CD8 + T-cell clone 6C5 was cultured in the presence of fractions (F1–F8) generated from the fractionation of the P1 peptide preparation (crude full length 9mer, LLYSFGTPT). T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + peptides at a 1 : 1 ratio for 5 hr. Lymphocytes were gated based on their forward and side scatter profile and then doublet exclusion was performed based on forward scatter height versus forward scatter width. T cells were then gated on CD3 + CD8 + cells and IFN- γ production was assessed through intracellular staining with anti-IFN- γ . Background (T-cells + T2) was subtracted from the data (mean ± SD, n = 3). Statistical analysis (unpaired two-tailed t -test, in comparison to P603 > 95% Pure) was carried out using GraphPad Prism . Significance is indicated by ****< 0·0001, ***0·0001–0·001, **0·001–0·01 and *0·01–0·05.

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Cell Culture, Staining, Fractionation, Solvent, Generated, Two Tailed Test, Comparison

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: Butylation of the tyrosine residue of p603 confers peptide reactivity. (a) Structure of modified peptide containing the alkylated residue Tyr(3- t Bu). Unmodified P603 (> 95% Pure) was reacted with methylpropene and trifluoroacetic acid (TFA), or Boc 2 O and TFA to induce t Bu groups on the tyrosine residue. (b) T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + P603 (> 95% Pure, 10 μg/ml), P603 (77% Pure, 10 μg/ml) or the two P603 modified peptides (di- tert -butyl dicarbonate and methylpropene) at a 1 : 1 ratio for 5 hr. Lymphocytes were gated based on their forward and side scatter profile and then doublet exclusion was performed based on forward scatter height versus forward scatter width. T cells were then gated on CD3 + CD8 + cells and IFN- γ production was assessed through intracellular staining with anti-IFN- γ . Background (T-cells + T2) were subtracted from the data (mean ± SD, n = 3). Statistical analysis (unpaired two-tailed t -test, in comparison to P603 > 95% Pure) was carried out using Graphpad Prism . Significance was indicated by ****< 0·0001.

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Residue, Modification, Cell Culture, Staining, Two Tailed Test, Comparison

Journal: Immunology

Article Title: CD8 + T-cell recognition of a synthetic epitope formed by t -butyl modification

doi: 10.1111/imm.12398

Figure Lengend Snippet: Activation of 6C5 is induced by t Bu P603. (a) and (b) Representative sample of flow cytometry analysis (three independent experiments) of intracellular staining of interferon- γ (IFN- γ ) and surface staining of CD107 α , respectively. T cells (1 × 10 5 /tube) were cultured in the presence of T2 or T2 + P603 (> 95% Pure, 10 μg/ml), P603 (77% Pure, 10 μg/ml) or P603 t Bu (> 98% Pure, 10 μg/ml) at a 1 : 1 ratio for 5 hr. IFN- γ production was assessed through intracellular staining with anti-IFN- γ and CD107 α expression through surface staining with anti-CD107 α . To facilitate gating, the cultures were also co-stained with anti-CD3 and anti-CD8 (mean ± SD of duplicates, n = 3). Statistical analysis (unpaired two-tailed t -test) was carried out using GraphPad Prism . (c) 5 × 10 5 T2 cells were pulsed with varying concentrations of P603 (77% Pure, 10 μg/ml to 1 × 10 −6 μg/ml) and P603 t Bu (> 98% Pure, 10 μg/ml to 1 × 10 −6 μg/ml). After 1 hr, unbound peptide was washed off and the pulsed T2 cells were cultured with 1 × 10 5 6C5 CD8 + T cells at a 1 : 1 ratio. Cell-free supernatant was harvested and tested for the presence of IFN- γ by ELISA (mean ± SD, n = 3). (d) 1 × 10 5 6C5 CD8 + T-cells were cultured at 1 : 1 ratio with T2 cells in the presence of varying concentrations of P603 (77% Pure, 10 μg/ml to 1 × 10 −6 μg/ml) and P603 t Bu (> 98% Pure, 10 μg/ml to 1 × 10 −6 μg/ml) for 5 hr. Lymphocytes were gated based on their forward and side scatter profile and then doublet exclusion was performed based on forward scatter height versus forward scatter width. T cells were then gated on CD3 + CD8 + cells and changes in the surface expression of CD107 α were determined through culturing the cells in the presences of anti-CD107 α (mean ± SD, n = 3). The EC 50 value was calculated using the fitted curve using Graphpad Prism .

Article Snippet: CD8 + T cells were immunomagnetically enriched from peripheral blood mononuclear cells using anti-human CD8 microbeads according to the manufacturer’s instructions (Miltenyi Biotec, Woking, UK).

Techniques: Activation Assay, Flow Cytometry, Staining, Cell Culture, Expressing, Two Tailed Test, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Disruptors of sestrin-MAPK interactions rejuvenate T cells and expand TCR specificity

doi: 10.1101/2024.05.17.594698

Figure Lengend Snippet: (A) Senescence-associated β-galactosidase expression in DOS-treated (DOS-juvenated) or untreated T sen . Cells were purified and cultured for one week in the presence of anti-CD3 (0.5 μg/mL) and rh-IL-2 (5 ng/mL), then stained to detect β-galactosidase activity. Representative image on the inverted phase-contrast microscope (left) and relative quantification (right, n = 8 donors). (B) Population doublings of human T sen (transduced with irrelevant scramble) and sestrin null CD4 + T sen (transduced by triple lentiviral depletion of sestrins) and cultured as in (A) (left). Donor-matched CD27 + CD28 + CD4 + T cells (herafter, T erl ) were cultured in parallel but activated with anti-CD3 and anti-CD28 ( n = 5 donors). Cells were cultured over two weeks with restimulation every 7 days. DOS-driven population doublings (right) were calculated as delta between DOS treated and DOS untreated T cells, with or without depletion of sestrins. (C) DOS-juvenation of human CD4 + T cells. Terminally differentiated effector memory CD45RA - CD28 - CD27 - CD4 + T cells (hereafter, T EM ) were purified and cultured over 20 days, as in (A). At day 1 (18 hours), 7, and 21 cell phenotypes were assessed by flow cytometry. Quantifications of rejuvenated stem like (CD28 + CD45RA + CCR7 + CD95 + CD62L + TCF1 + ) among human CD4 + T cells are shown (left; n = 5 donors). Decay of T EM and CD28 - CD27 - CD45RA + CD4 + T cells (hereafter, T EMRA ) undergoing rejuvenation is shown (right). (D) Adoptive transfer of DOS-juvenated T cells, experimental design. Donor T cells were derived from twenty-month-old mice 15 days after Fluad vaccination with or without DOS treatment (0.1 mg/Kg throughout), labeled with Cell Trace Violet (CTV) dye or congenic CD45.1 tracking, then transferred into young naïve CD45.2 recipients (3 months). In parallel, young mice (3 months) were used as young donor control. Recipient animals were rested for 28 days, then analysed for donor T cell persistence and maintenance of stem phenotype after transfer. (E) Maintenance of donor DOS-juvenated CD45.1 CD4 + T cells, their aged-matched controls, and that of young donor T cells, 28 days after transfer (day 43) in recipient mouse lymph nodes. Representative flow cytometry plots and poled data ( n = 5 mice per group) are shown. (F) Assessment of mouse T cell memory programs in stem like transferred T cells (among CD45.1 CD44 - CD62L + CD95 + CD4 + T cells) and terminally differentiated cells (TE, among CD45.1 CD44 - CD62L - CD4 + T cells) following adoptive transfer as in (D) ( n = 5 mice). (G) IL7R gene expression and (H) CD95 and Sca-1 mean fluorescent intensity (MFI; throughout) in stem cells derived from CD4 + CD45.1 + transferred stem T cells among lymph nodes of recipient CD45.2 mice, 28 days after transfer ( n = 5 mice). (I) Representative plots of CD45.1 transferred cells in quiescent state before and after transfer assessed by cycle related intra-nuclear Ki67 staining. Representative of n = 5 mice per group. (J) G1 (Ki67 + ) to G0 (Ki67 - ) transition in stem like CD45.1 CD4 + T cells before and after adoptive transfer as indicated ( n = 5 mice per group). (K) Assessment of T cell longevity following DOS-juvenation. Cells from lymph nodes were stained using the Annexin-PI Apoptosis detection Kit 28 days after transfer. Representative FACS plot (left) and quantification of dead CD45.1 + CD4 + transferred T cells (right). (L) DOS-juvenated T cell maintenance, in vivo model. In (A and B, right) two tailed paired T test was used. In (B, left-C, E-H and J-K) one-way Anova with Bonferroni post-correction for multiple comparisons was used, *p<0,05, **P<0,01; ***P<0,001; ****P<0,0001. Error bars indicate SEM.

Article Snippet: Primary human CD4 + T cells were isolated using CD4 MicroBeads (130-045-101; Miltenyi); primary human senescent CD4 + CD27 - CD28 - T cells (thereafter T sen ) were isolated using CD4 + T Cell Isolation Kit (130-096-533; Miltenyi) followed by depletion of CD27 + and CD28 + T cells using CD27 MicroBeads (130-051-601; Miltenyi) and CD28 MicroBead Kit (130-093-247; Miltenyi).

Techniques: Expressing, Purification, Cell Culture, Staining, Activity Assay, Microscopy, Quantitative Proteomics, Transduction, Flow Cytometry, Adoptive Transfer Assay, Derivative Assay, Labeling, Control, Gene Expression, In Vivo, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: Healthy human donor PBMCs were inoculated with mock or VZV infected ARPE-19 epithelial cells for 2 days then analysed for infection by flow cytometry. (A) Representative flow cytometry plots of mock or VZV-S infection, examining surface VZV gE:gI expression on live T cells (CD3 + CD56 – ), CD3 + CD56 + lymphocytes, and NK cells (CD3 – CD56 + ). (B) Frequencies of live gE:gI + lymphocytes in total (shaded), compared to specific populations: T cells, CD3 + CD56 + lymphocytes, and NK cells (n = 19). Symbols represent individual donors consistent across lymphocyte populations, and bars indicate mean. Statistical analysis was performed between specific lymphocyte populations. **p < 0.01, ****p < 0.0001 (RM one-way ANOVA with the Greenhouse-Geisser correction and Tukey’s multiple comparisons test). (C) Representative flow cytometry plots of vOka infection, examining surface gE:gI expression on live T cells (CD3 + CD56 – ), CD3 + CD56 + lymphocytes, and NK cells (CD3 – CD56 + ) (n = 3).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Expressing

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: (A) Healthy human donor PBMCs were infected with VZV by cell-associated infection with or without IL-2 (200 U/ml) for 2 days, then analysed by flow cytometry. Plots show surface VZV gE:gI expression from one representative donor and graphs show frequency of live gE:gI + NK cells (CD3 – CD56 + ) (top panels), CD3 + CD56 + lymphocytes (middle panels), and T cells (CD3 + CD56 – ) (bottom panels). Symbols represent individual donors consistent across lymphocyte populations, and bars indicate mean (n = 8). ***p < 0.001, ****p < 0.0001 (two-tailed paired t test). (B & C) Healthy human donor CD56 + -selected lymphocytes were infected with VZV by cell-associated infection with or without IL-2 (200 U/ml) for 2 days, then analysed by flow cytometry. Plots show surface gE:gI expression from one representative donor and graphs show frequency of live gE:gI + NK cells (B) or CD3 + CD56 + lymphocytes (C). Symbols represent individual donors, consistent across (B & C) (n = 7). *p < 0.05 (two-tailed Wilcoxon matched-pairs signed rank test).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Expressing, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: NK cells (CD3 – CD56 + ) were FACS sorted from healthy human donor CD56 + -selected lymphocytes following mock or VZV infection for 1 day. (A & B) Staining by IFA of sorted VZV cultured (left panels) or mock cultured (right panels) NK cells for IE63 (A), pORF29 (B) or respective isotype control, with DAPI (n = 3). (C) Sorted VZV cultured NK cells were added to ARPE-19 epithelial cell monolayers. Four days later monolayers were fixed and infectious centres detected with IFA by staining for IE63 and gE:gI, with DAPI. One representative experiment of five is shown.

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Staining, Cell Culture, Control

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: (A) Healthy human donor PBMCs were infected with VZV for 2 days then analysed for infection by flow cytometry. Plots show gating strategy for CD56 bright and CD56 dim NK cells (CD3 – CD56 + ) (left panel), with respective surface VZV gE:gI expression (right panels) from one representative donor (n = >7). (B & C) CD3 – CD56 bright (B) and CD3 – CD56 dim (C) NK cells were isolated from healthy human donor PBMCs by FACS sorting and subsequently infected with VZV for 2 days before analysis by flow cytometry. Plots show surface gE:gI expression from one representative donor (n = 2).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Expressing, Isolation

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: Healthy human donor PBMCs were mock or VZV infected with or without IL-2 (200 U/ml) for 2 days then analysed by flow cytometry. (A) Diagram describes gating strategy and tSNE analysis workflow for samples shown in (B & C). (B & C) tSNE plots show marker expression levels for single parameters on individual cells in the tSNE map for mock and VZV cultured NK cells after 2 days, either untreated (B) or in the presence of IL-2 (C). Arrowheads indicate the CD56 bright NK cell subset, and the outlined population indicates the localisation of VZV + NK cells. One representative experiment of three is shown. (D & E) Plots show CD57 expression between mock and VZV cultured NK cells (D) and between bystander and VZV + NK cells (E), from one representative donor. Graphs show respective frequencies of CD57 + NK cells when untreated or with IL-2 (shaded) for four donors. Bars indicate mean. (F) Histograms show CD16 expression for mock, bystander and VZV + NK cells from one representative donor. Graph shows frequency of CD16 + NK cells when untreated or with IL-2 (shaded) for six donors. Bars indicate mean. *p < 0.05, **p < 0.01, ***p < 0.001 (Friedman test with Dunn’s multiple comparisons test).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Marker, Expressing, Cell Culture

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: CD3 – CD56 + CD57 – NK cells and CD3 – CD56 + CD57 bright NK cells were isolated from healthy human donor PBMCs by FACS sorting and subsequently mock or VZV infected with or without IL-2 (200 U/ml) for 2 days before analysis by flow cytometry. (A) Diagram describes experimental design of isolating NK cells on CD57 expression, then infecting, and subsequently analysing for infection and phenotype changes. (B) Plots show surface VZV gE:gI expression between subsets from one representative donor. Graph shows frequency of VZV + NK cell subsets when untreated or with IL-2 (shaded) for three donors. Bars indicate mean. (C) Plots show subsequent CD57 expression between mock, bystander and VZV + CD57 – NK cells (left panels) and CD57 versus gE:gI expression for VZV cultured CD57 – NK cells (middle panels), from one representative donor. Graphs show frequency of CD57 expression on mock, bystander and VZV + CD57 – NK cells for three donors. Bars indicate mean. *p < 0.05 (two-tailed paired t test). (D) Histograms show CD16 expression for mock, bystander and VZV + CD57 – NK cells (left panel) and CD57 bright NK cells (right panel) for one representative donor (n = 3).

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Isolation, Infection, Flow Cytometry, Expressing, Cell Culture, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Varicella zoster virus productively infects human natural killer cells and manipulates phenotype

doi: 10.1371/journal.ppat.1006999

Figure Lengend Snippet: Healthy human donor PBMCs were mock or VZV infected with or without IL-2 (200 U/ml) for 2 days then analysed by flow cytometry. (Left panels) Representative plots show CCR4 (A) or CLA (B) expression against CD56 expression for mock, bystander and VZV + NK cells. (Right panels) Representative plots show CCR4 (A) or CLA (B) expression versus VZV gE:gI expression for VZV cultured NK cells. Data are representative of five donors.

Article Snippet: For some experiments, where specified, CD56 + lymphocytes were isolated from PBMC by MACS positive selection using CD56 MicroBeads, according to manufacturer’s protocol (Miltenyi Biotech).

Techniques: Infection, Flow Cytometry, Expressing, Cell Culture

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

Article Title: Intratumoral activation of 41BB co-stimulatory signals enhances CD8 T cell expansion and modulates tumor-infiltrating myeloid cells

doi: 10.4049/jimmunol.2000759

Figure Lengend Snippet: Intratumoral treatment with agonistic 41BB antibodies increases CD8 T cell infiltration. A and B, Mass of MC38 and Panc02-ZsGOVA tumors harvested 7 days after initial α41BB treatment. C and D, Frequency of CD4 and CD8 TILs in MC38 and Panc02-ZsGOVA tumors 7 days after initial α41BB treatment. E, Mice with MC38 tumors were treated with isotype or α41BB antibodies in combination with CD8 depleting antibodies. Tumor growth is shown (n=5 mice per group). F, TILs were isolated from MC38 tumors treated with intratumoral isotype or α41BB antibodies. TILs were cultured with immobilized anti-CD3 antibody or co-cultured with irradiated MC38 or B16 tumor lines for 48hrs. IFN-γ was measured from supernatants. One of two representative experiments are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: For surface staining of murine specimens, cells were stained in FACS buffer with the following antibodies: CD3 (145–2C11), CD4 (GK1.5), CD8 (53–6.7), CD11b (M1/70), Ly6G (1A8), Ly6C (HK1.4), F4/80 (BM8), CD11c (N418), MHCII (M5/114.15.2), CD80 (16–10A1), CD86 (GL-1), PD-1 (29F.1A12) (all from BioLegend); 41BB (17B5–1H1, Miltenyi Biotec).

Techniques: Isolation, Cell Culture, Irradiation

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

Article Title: Intratumoral activation of 41BB co-stimulatory signals enhances CD8 T cell expansion and modulates tumor-infiltrating myeloid cells

doi: 10.4049/jimmunol.2000759

Figure Lengend Snippet: 41BBL expression on APCs alters the capacity to prime TILs. TILs from a melanoma patient were cultured with αCD3, autologous tumor cells at a 1:1 ratio, or tumor-lysate pulsed 41BBL APCs at a 1:10 ratio for 72hrs. A and B, Heatmap representing cytokine abundance in supernatants from cell cultures were collected at 72hrs. Numerical values are indicated for each parameter with its respective condition. Cytokine production by TIL stimulated with αCD3 +/− urelumab (A). Cytokines in TIL-tumor co-cultures +/− α41BB or MHC-I blocking antibodies (B). C, 41BBL APCs were generated and then pulsed with autologous tumor lysate in the presence of GMCSF. Pulsed APCs were then seeded in culture wells with or without α41BB. D, Cytokines were measured in the supernatants of TIL-APC co-cultures incubated with α41BB and/or α 41BBL. TIL only condition is the same data from (B); APCs only from (C). Statistics are indicated for cytokines that are higher than both TILs alone and APCs alone conditions. E, Fold change in cytokine induction vs. TIL+APCs in co-cultures from (D). Dotted line represents the basal induction of cytokines in TIL-APC co-cultures. F, TIL proliferation in co-cultures was determined in the final 18hrs of the culture by 3H thymidine incorporation in the presence of soluble α41BB and/or soluble α41BBL. Dotted line represents basal TIL proliferation without additional stimulation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Significance was determined by two-tailed t-test or 2-way ANOVA with Dunnett’s multiple comparisons. ND = not detected.

Article Snippet: For surface staining of murine specimens, cells were stained in FACS buffer with the following antibodies: CD3 (145–2C11), CD4 (GK1.5), CD8 (53–6.7), CD11b (M1/70), Ly6G (1A8), Ly6C (HK1.4), F4/80 (BM8), CD11c (N418), MHCII (M5/114.15.2), CD80 (16–10A1), CD86 (GL-1), PD-1 (29F.1A12) (all from BioLegend); 41BB (17B5–1H1, Miltenyi Biotec).

Techniques: Expressing, Cell Culture, Blocking Assay, Generated, Incubation, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Heatmap of IRF family members expression in NK cells throughout the course of MCMV infection represented as z-score of log2 normalized counts based on RNA-seq. b. Representative histogram of IRF4 expression in WT and Irf4−/− NK cells on day 2 PI (n = 4 biological replicates). c. Tracks (top) show chromatin accessibility dynamics of the Irf4 locus in Ly49H+ NK cells at days 0, 2, 4, and 7 PI as assessed by ATAC-seq. Graphs (bottom) show normalized counts for each peak indicated. d. Tables of enriched known motifs of highly accessible regions (log2FC > 1 & padj < 0.05) on day 4 PI versus day 2 PI from ATAC-seq data of Ly49H+ NK cells after MCMV infection. e. Heatmap of motif score from de novo motif analysis on highly accessible regions (log2FC > 1 & padj < 0.05) during day 0, 2, 4, and 7 transition based on ATAC-seq data of Ly49H+ NK cells after MCMV infection. f. IRF4 expression of sorted naïve splenic NK cells after an overnight stimulation with the indicated stimuli. IRF4 induction is displayed as fold change of IRF4 MFI over unstimulated condition (n = 6 biological replicates per condition). Two-way ANOVA test adjusted for multiple comparisons was used for statistical analysis. g. Representative histogram of IRF4 expression upon indicated stimulation gated on human CD56bright NK cells. Data are presented as paired fold change of IRF4 MFI compared to unstimulated condition (n = 8 donors per condition). Two-way ANOVA test adjusted for multiple comparisons was used for statistical analysis. h. UMAP embedding of scATAC-seq data from in vitro stimulated human NK cells (sorted on CD3−CD14−CD19−CD7+NKG2C+) from HCMV− donors. i. Coverage plot of the IRF4 locus from in vitro stimulated human NK cells as in (H).

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Control, Expressing, Infection, RNA Sequencing, In Vitro

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Scatter-plot comparing the expression of IRF family members that are differentially expressed on days 2 and 4 PI versus day 0 in Ly49H+ NK cells by RNA-seq analysis. Points highlighted in red and blue indicate genes that are upregulated and downregulated, respectively, on day 2 versus day 0 and day 4 versus day 0 (Padj < 0.05). b, Kinetics of RNA-seq normalized reads for Irf4 transcripts in Ly49H+ NK cells throughout the course of MCMV infection (n = 2 biological replicates per time point). c, Representative flow cytometric histogram of IRF4 expression in splenic Ly49H+ NK cells in MCMV-infected mice (left) and fold change of IRF4 mean fluorescent intensity (MFI) compared to uninfected mice (right) (n = 3 biological replicates for day 0, 2 and 3 and n = 2 biological replicates for day 4). d, UMAP embedding of scATAC-seq data from human NK cells of HCMV+ or HCMV− healthy donors. e, Coverage plot of the IRF4 locus from defined human NK cell subsets as in d. f, RNA-seq reads for IRF4 expression from human NK cell subsets represented as fragments per kilobase of transcript per million mapped reads (FPKM) (n = 5). CD56bright (CD56brightCD16dim), early CD56dim (CD56dimNKG2A+KIR−CD57−), CD56dim (CD56dimNKG2A−self-KIR+CD57+), adaptive NKG2C+ (CD56dimCD16+NKG2C+CD7lowNKp30lowCD57+). g, Representative histogram of IRF4 expression of sorted naive splenic NK cells after an overnight stimulation with the indicated stimuli. IRF4 induction is displayed as FC of IRF4 MFI over unstimulated condition (n = 3 per group). h, Volcano plot of RNA-seq data from human NK cells stimulated with HCMV UL-40 peptide + IL-12/18 versus control peptide. Red and blue points represent DEGs (Padj < 0.05 and |log2FC| > 0.5) in stimulated and control conditions, respectively. Data are represented as mean ± s.e.m. and are representative of, or pooled from, at least two independent experiments. A two-tailed unpaired t-test was used for g. Statistical analysis was not derived from groups with n < 3.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, RNA Sequencing, Infection, Control, Two Tailed Test, Derivative Assay

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, GSEA pathway analysis of DEGs between WT and Irf4−/−Ly49H+ cells on day 7 PI, from scRNA-seq experiment in Fig. 4. b, Representative histograms of CD98 expression between naive splenic WT and Irf4−/−Ly49H+ NK cells (dashed lines) or day 5 PI (solid lines). Data are represented as FC gMFI from WT cells taken from day 5 MCMV-infected mice (n = 8 biological replicates). c, Representative histograms of CD71 expression between naive splenic WT and Irf4−/−Ly49H+ NK cells (dashed lines) or day 5 PI (solid lines). Data are represented as FC gMFI from WT cells taken from day 5 MCMV-infected mice (n = 8 biological replicates). d, Representative histogram and gMFI of kynurenine uptake by splenic WT or Irf4−/−Ly49H+ NK cells taken from day 5 PI. Kyn + lysine (Lys) was added as a non-competitive control and Kyn + leucine (Leu) acted as a competitive control (n = 4 biological replicates in each condition). e, Representative histogram and gMFI of transferrin uptake assay from naive or day 5 PI splenic WT or Irf4−/−Ly49H+ NK cells (n = 11 biological replicates). Data are represented as mean ± s.e.m. and are representative of or pooled from at least two independent experiments. A two-tailed paired t-test was performed for b–e and adjusted for multiple comparisons for d.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Infection, Control, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, b Representative histograms and quantification of IFN-γ production (A) and LAMP-1 (also known as CD107a) (B) from either naive WT (gray) or Irf4−/− (orange) NK cells upon in vitro stimulation with nothing, IL-12 + IL-18, PMA + Ionomycin, or PMA + Ionomycin + IL-12 for 3 hours. c–f. Representative histograms (upper panels) and quantifications (bottom panels) of CD25, Gzmb, CD69, and IFN-γ expression gated on Ly49H+ NK cells from MCMV-infected WT:Irf4−/− mBMC on day 2 PI. Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. n = 3 biological replicates per group. Paired two-tailed t-test was performed unless stated otherwise.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: In Vitro, Expressing, Infection, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a–c. Number of NK cells in blood (A), liver (B), and spleen (C) of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 6) mice at 8 weeks old. d–f. Maturation status based on the expression of CD27 and CD11b of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 5 for blood, and n = 6 for liver and spleen) mice in the blood (D), liver (E), and spleen (F) at 8 weeks old. g. Percentage of Ly49H+ NK cells within NK cells (Lineage-NK1.1+ CD49b+) of WT (n = 8), Irf4+/− (n = 4), or Irf4−/− (n = 6) mice at 8 weeks old. h. Experimental schematic of WT:Irf4−/− mixed bone-marrow chimera (mBMC) generation. i–k. Percentage of NK cells of WT or Irf4−/− mice from WT:Irf4−/− mBMC in the blood (n = 60 biological replicates) (I) and spleen (n = 21 biological replicates) ( J) after 8 weeks (I-J), 16 weeks (n = 3 biological replicates) or 6 months (n = 5 biological replicates) post-transplant (K). l. Representative of flow plots of CD62L versus CD27 of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC in the blood and spleen 8 weeks post-transplant. Data are represented as percentage of each subset within total NK cells of each genotype (n = 22 biological replicates in blood, and n = 13 biological replicates in spleen). m. Representative of flow plots of CD11b versus CD27 of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC in the blood and spleen 8 weeks post-transplant. Data are represented as percentage of each subset within total NK cells of each genotype (n = 22 biological replicates in blood, and n = 16 biological replicates in spleen). n. Histogram and percentage quantification of Ly49H+ NK cells within splenic NK cells of either WT or Irf4−/− NK cells from WT:Irf4−/− mBMC (n = 24 biological replicates). Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. Unpaired (A-G) and paired (I-N) two-tailed t-tests were performed.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Experimental schematic of adoptive transfer of WT and Irf4−/− NK cells into Rag2−/− Il2rg−/− mice. b. Fold change chimerism between WT and Irf4−/− NK cells on day 7 over day 0 (pre-transfer) in blood, liver, and spleen (n = 3 biological replicates in blood, and n = 6 biological replicates in liver and spleen). c. Representative histogram of IRF4 expression between transferred WT and Irf4−/− NK cells on day 7 post-transfer into Rag2−/− Il2rg−/−. d. Representative flow plots of CD27 and CD11b expression between WT and Irf4−/− NK cells on day 7 post-transfer. Data are represented as percentage of each subset within WT or Irf4−/− NK cells in indicated tissues (n = 3 biological replicates). e. Histogram of CD122 and quantification of CD122 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). f. Histogram of CD132 and quantification of CD132 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). g. Histogram of pSTAT5 and quantification of pSTAT5 MFI between WT and Irf4−/− NK cells on day 7 post-transfer in the spleen (n = 9 biological replicates). H-J. h–j. Quantification of Ki-67 staining in the blood and spleen between WT and Irf4−/− NK cells (H, n = 5 biological replicates per group), BIM and BCL2 MFI from splenic NK cells (I-J, n = 9 biological replicates). Data are represented as mean ± SEM and are representative of or pooled from at least two independent experiments. Paired two-tailed t-test was performed unless stated otherwise.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Adoptive Transfer Assay, Expressing, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Kaplan–Meier survival curves from Rag2−/− Il2rg−/− mice transferred with WT Ly49H+ NK, Irf4−/−Ly49H+ NK or no NK cells (n as indicated). b, Percentage of Ly49H+ NK cells from adoptive co-transfer of WT (CD45.1) and Irf4−/− (CD45.2) NK cells into Ly49H-deficient mice from the blood throughout the course of MCMV infection (left) and percent chimerism of WT versus Irf4−/− transferred Ly49H+ NK cells (right) (n = 4 biological replicates). c, Representative flow plots gated on transferred WT (CD45.1) and Irf4−/− (CD45.2) Ly49H+ NK cells on day 7 in different tissues (left) and the percent chimerism of WT and Irf4−/− NK cells within transferred Ly49H+ NK cells between different tissues (n = 4 biological replicates per group). d, Representative histogram of CTV dilution on day 0 and day 4 PI gated on transferred Ly49H+ NK cells (left). Dashed gray and orange lines represent WT and Irf4−/− NK cells on day 0, respectively; solid gray and orange lines represent WT and Irf4−/− NK cells on day 4 PI, respectively. Percentage of transferred NK cells that have divided at least once (n = 5 biological replicates per group) (right). e, Representative histogram of BIM expression on day 5 PI from splenic WT or Irf4−/−Ly49H+ NK cells (left) and BIM gMFI (right) (n = 4 biological replicates). gMFI, geometric mean fluorescence intensity. f, Percentage of FLICA+ (marking activated caspase) WT or Irf4−/−Ly49H+ NK cells taken from day 5 PI WT:Irf4−/− mBMC splenocytes upon ex vivo treatment with ABT199 (BCL2 inhibitor) (n = 3 biological replicates in each condition). Data are represented as mean ± s.e.m. and are representative of, or pooled from, at least two independent experiments. For statistical testing, a log-rank test was used for a and a two-tailed paired t-test was used for b–f and adjusted for multiple comparisons for b and c.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, Expressing, Fluorescence, Ex Vivo, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Representative flow plots showing CD62L and CD27 expression of transferred WT or Irf4−/−Ly49H+ NK cells on day 7 PI (left). Percentage of NK cell subset based on CD62L and CD27 expression of transferred cells on day 7 PI in the blood, liver and spleen (right) (n = 5 biological replicates in blood and spleen and n = 4 biological replicates in liver). b, Representative histogram and percentages of Ly6C, CX3CR1 and KLRG1 of transferred WT or Irf4−/− Ly49H+ NK cells on day 7 PI from spleens of MCMV-infected mice (n = 5 per group). c, Fold change of chimerism between splenic WT and Irf4−/−Ly49H+ NK cells upon infection of WT:Irf4−/− mBMC with MCMV from day 0, 2, 4, 5 and 7 PI (n = 6 biological replicates for day 0 and 7, n = 5 biological replicates for day 2, n = 7 and 8 biological replicates for day 4 and 5, respectively). d, Kinetics of different subsets of splenic WT and Irf4−/−Ly49H+ NK cells from WT:Irf4−/− mBMC during MCMV infection setting based on CD62L and CD27 expression from day 0 to 7 PI (n = 6 biological replicates for day 0 and 7, n = 7 biological replicates for day 2 and n = 8 biological replicates for day 4 and 5, respectively). Data are represented as mean ± s.e.m. and are representative of or pooled from at least two independent experiments. A two-tailed paired t-test was performed for b–d and adjusted for multiple comparisons for c and d.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Expressing, Infection, Two Tailed Test

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, WT and Irf4−/− Ly49H+ NK cells were sorted on day 0, 2, 4 and 7 following MCMV infection and single-cell RNA-sequencing performed on multiplexed samples. UMAP embedding of scRNA-seq colored by Louvain clusters (kn = 30). b, Top 50 DEGs between WT and Irf4−/−Ly49H+ NK cells during MCMV shown as z score of normalized reads. c, PAGA network analysis based on scRNA-seq data from WT versus Irf4−/−Ly49H+ NK cells scRNA-seq. d, UMAP embedding of WT and Irf4−/−Ly49H+ NK cells on day 7 from clusters 1 and 3 in a. e, Volcano plot of scRNA-seq as determined by MAST between WT and Irf4−/−Ly49H+ NK cells on day 7 PI. Blue and yellow points represent significant genes (FDR < 0.05) that are upregulated in WT or Irf4−/−Ly49H+ NK cells, respectively. Highlighted genes are effector genes and/or factors known to be important for NK cell differentiation/maturation. f, Dot plot of scRNA-seq for maturation markers between WT and Irf4−/−Ly49H+ NK cells on day 7 PI shown as mean normalized expression and percent of cell fraction within the group. g, UMAP plot from d as MPEC or SLEC gene signature module scores.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, RNA Sequencing, Cell Differentiation, Expressing

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. UMAP embedding of WT (left) or Irf4−/− (right) of Ly49H+ NK cells colored by genotypes and days post-infection. b. Proportion of each genotype from each time points per Louvain clusters. c. Volcano plot of scRNA-seq as determined by MAST between WT and Irf4−/− Ly49H+ NK cells on day 0, 2, 4 and 7 PI. Blue and yellow points represent significant genes (FDR < 0.05) that are upregulated in WT or Irf4−/− Ly49H+ NK cells, respectively. d. Number of differentially expressed genes between WT or Irf4−/− Ly49H+ NK cells from each time point as identified by MAST. e. Violin plot quantification of normalized reads for MYC transcript by scRNA-seq between WT and Irf4−/− Ly49H+ NK cells. f. Representative histogram of Myc protein by flow cytometry between WT (gray) and Irf4−/− (orange) Ly49H+ NK cells on day 5 PI. g. Violin plot quantification of MAGIC-imputed reads for CD71 (Tfrc), IRP1 (Aco1), and IRP2 (Ireb2) on day 7 PI between WT (blue) and Irf4−/− (yellow) Ly49H+ NK cells by scRNA-seq.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Infection, Flow Cytometry

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a, Normalized count heat map of IRF4 binding upon anti-NK1.1 + cytokines (IFN-α + IL-12/18 + IL-2/15) stimulation or IL-15 alone at +0.5 kb from the peak summit (n = 12,344 peaks). b, IGV tracks showing IRF4-binding signal between unstimulated (IL-15 alone, gray) or stimulated (anti-NK1.1 + cytokines, orange) conditions in Bcl2l11, Sell, Tfrc and Ireb2 loci. Regions highlighted in dashed boxes represent called IRF4 peaks by MACS2 in stimulated condition. c, Top: Venn diagram comparing IRF4-bound genes in stimulated condition and DEGs between WT versus Irf4−/−Ly49H+ NK cells on day 7 PI from scRNA-seq. Bottom: distribution of IRF4-bound DEGs between WT versus Irf4−/−Ly49H+ NK shown as fold change as analyzed by MAST. d, Proportion of IRF4 genome-wide occupancy within the exon, intergenic, intronic and promoter regions. e, Enriched de novo motifs found by HOMER on IRF4-bound regions upon stimulation. P values were calculated by HOMER (Methods). f, Metacoverage of IRF4-bound accessible regions that contain the indicated motifs in Ly49H+ NK cells upon MCMV infection as assessed by ATAC-seq. Data are shown as median of normalized counts. g, Top: the z score of DEGs that contain AICE motifs in Ly49H+ NK cells during MCMV infection. Bottom: average of scaled log-normalized counts of AICE-motif-containing genes enriched in either WT or Irf4−/−Ly49H+ NK cells (Methods) throughout the course of MCMV. Adjusted P values (Padj) were calculated by ‘geseca’ function of the fgsea package (Methods).

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques: Binding Assay, Genome Wide, Infection

Journal: Nature immunology

Article Title: Control of nutrient uptake by IRF4 orchestrates innate immune memory

doi: 10.1038/s41590-023-01620-z

Figure Lengend Snippet: a. Gating strategy for sorting WT or Irf4−/− Ly49H+ NK cells. b. Gating strategy for phenotypic analysis of Ly49H+ NK cells after lymphocytes, singlets and live cells cleanup (as depicted in Extended Data Fig. 7a). c. Gating strategy for human NK cells.

Article Snippet: IRF4 chromatin immunoprecipitation by sequencing Splenic WT NK cells (NK1.1 + CD49b + ) were expanded ex vivo for 2 weeks in complete IMDM (10% FBS, 1% pen/strep, 1× BME, 1× MEM-NAA, 1× HEPES) in the presence of 50 ng ml −1 of human IL-15 (Miltenyi, cat. no. 130–095-766).

Techniques:

Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: The OPN-i deficient T FH and T FR phenotype is cell-intrinsic. a, Flow cytometry of donor OT-II CD4 + T cells from spleens of Rag2 −/− Prf1 −/− hosts transferred with naïve OT-II, OT-II OPN-KO and OT-II OPN-i-KI CD4 + T cells along with wild-type B cells, followed by immunization with NP 13 -OVA in CFA and analysis 10 d later. Numbers indicate percent T FH (Foxp3 − PD1 + CXCR5 + CD4 + ) and GC B (Fas + GL7 + B220 + ) cells. b, Serum titers of total (NP 23 ) NP-specific IgG and IgG1 from recipient mice in a ( n = 6 per group). c, Flow cytometry of donor Treg from spleens of Tcr α −/− hosts transferred with sorted CD45.2 + Treg (CD25 hi CD44 int CXCR5 − CD4 + ) from the indicated mice and CD45.1 + wild-type naïve CD4 + T cells (CD25 − GITR − CD44 lo CD62L hi ) at a ratio of 1:2, followed by immunization with NP 26 -KLH in CFA and analysis 10 d later. Numbers indicate percent T FR (CD45.1 − CD44 + Foxp3 + PD1 + CXCR5 + CD4 + ) and GC B (Fas + GL7 + B220 + ) cells. d, Frequency of T FR and GC B cells in c ( n = 5 per group). e, Titers of total (NP 23 ) and high-affinity (NP 4 ) NP-specific IgG at d14 in immunized Rag2 −/− Prf1 −/− recipients of OPN-i-KI or OPN-KO T FH cells (5 × 10 4 ) and/or OPN-i-KI or OPN-KO T FR cells (2.5 × 10 4 ) (sorted as in ) and wild-type GL-7 − B cells (1 × 10 5 ) from KLH-immunized mice. All recipients ( n = 4 per group) were immunized with NP 26 -KLH in CFA. f, Donor GC B cells from spleens of recipients in e (in same order from left to right) at d22. g, Titers of NP-specific total IgG at d11 in immunized Rag2 −/− Prf1 −/− mice ( n = 3 per transfer) given OPN-i-KI or OPN-KO T FH cells and/or OPN-i-KI or OPN KO T FR cells (2.5 × 10 4 ) at different ratios and wild-type GL-7 − B cells (1 × 10 5 ) from KLH-immunized mice. Data are representative of three ( a–b ) and two ( c–g ) independent experiments. * P < 0.05 and ** P < 0.01 (unpaired two-tailed Student’s t-test; error bars, mean ± s.e.m).

Article Snippet: CD62L − CD4 + T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4 + CD62L + T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining.

Techniques: Flow Cytometry, Two Tailed Test

Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: ICOS co-stimulation promotes an interaction between OPN-i and p85α. a, Quantitative RT-PCR analysis of Spp1 mRNA in naïve CD4 + T cells from B6 mice stimulated with anti-CD3 and anti-CD28 for 2 d, followed by resting overnight before 20 m incubation with the indicated Abs and cross-linking with goat anti-hamster Ab for 8 h or 24 h. Spp1 expression was normalized to the Rps18 control and results are presented relative to isotype-matched hamster IgG-treated cells at 8 h, set as 1. * P < 0.05 (unpaired two-tailed Student’s t-test; error bars, mean ± s.e.m of quadruplicates). b, Immunoblot analysis of OPN and actin of CD4 + T cells in a after 12 h cross-linking. c, Immunoassay of lysates of purified CD62L − CD4 + T cells from Icos −/− and wild-type mice 2 or 3 d after intraperitoneal injection with 100 μg KLH in CFA, probed with anti-Bcl-6, anti-OPN and anti-actin. d, Immunoblot analysis of lysates of sorted V β 5 + CD25 − CD44 hi GITR − CD4 + effector and CD25 + CD44 hi GITR + CD4 + regulatory T cells from pooled OT-II ( n = 10) or OT-II Icos −/− mice ( n = 15) 8 d post-immunization with OVA in CFA, probed as in c. e, Immunoassay of lysates of 293T cells transfected with plasmids encoding Flag-tagged p85α (Flag-p85α) and increasing concentrations of OPN-i, assessed by immunoprecipitation (IP) with anti-Flag and immunoblot analysis with anti-Flag and anti-OPN. f, Immunoassay of lysates of purified naïve CD4 + T cells from OPN-KO and wild-type mice stimulated with anti-CD3 and anti-CD28 for 2 d (left); or CD44 + CD4 + T cells from OT-II mice 4 d post-immunization with OVA in CFA (right), followed by resting and re-stimulation with anti-CD3 and/or anti-ICOS (as in a ) for 12 h, and assessed by IP with anti-p85α and immunoblot analysis with anti-p85α and anti-OPN. Data represent two ( a, c, d ) and three ( b, e, f ) independent experiments.

Article Snippet: CD62L − CD4 + T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4 + CD62L + T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining.

Techniques: Quantitative RT-PCR, Incubation, Expressing, Control, Two Tailed Test, Western Blot, Purification, Injection, Transfection, Immunoprecipitation

Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: p85α chaperones nuclear translocation of OPN-i. a, Immunoassay of lysates of 293T cells transfected with plasmids encoding Flag-p85α and OPN-i wild-type or OPN-i Y166F (YF) mutant, assessed by immunoprecipitation with anti-Flag and immunoblot analysis with anti-Flag and anti-OPN. Input, immunoblot analysis of an aliquot of cell lysates without IP. b, Confocal microscopy of OPN and Bcl-6 expression by CD44 + CD4 + T cells from OPN-i-KI mice 3 d post-injection with KLH in CFA, followed by cross-linking with anti-ICOS Ab (as in ) in vitro . Cells were counterstained with DNA-intercalating dye DAPI to trace nuclear perimeters. About 25–30 cells stained with Bcl-6 were further analyzed for localization of OPN protein. Right, fluorescence intensity expressed as the mean ratio of nuclear (Nuc) to cytoplasmic (Cyt) fluorescence pixel intensity ( n = 25–30 cells). c, Immunoblot analysis of nuclear fractions of OPN-i-KI CD62L − CD4 + T cells treated with anti-CD3 and/or anti-ICOS Abs (as in ), probed with anti-OPN, anti-Lamin B1 and anti-tubulin (to validate the integrity of nuclear separation). d , Immunoblot analysis of nuclear and cytosolic fractions of 293T cells transfected with plasmids encoding OPN-i wild-type or OPN-i Y166F mutant and increasing concentrations of Flag-p85α, probed with Abs, as indicated. e, Confocal microscopy of OPN and Bcl-6 expression by CD62L − CD4 + T cells from p85α WT or KO mice. Cell treatment and analysis as in a . Original magnification ( b, e ), 600×. * P < 0.05 and *** P < 0.001, Mann-Whitney test (error bars, mean ± s.e.m) (b, e ). All results are representative of at least two independent experiments.

Article Snippet: CD62L − CD4 + T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4 + CD62L + T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining.

Techniques: Translocation Assay, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Confocal Microscopy, Expressing, Injection, In Vitro, Staining, Fluorescence, MANN-WHITNEY

Journal: Nature immunology

Article Title: A p85α–osteopontin axis couples the ICOS receptor to sustained Bcl-6 expression by follicular helper and regulatory T cells

doi: 10.1038/ni.3050

Figure Lengend Snippet: Intranuclear OPN-i interacts with and stabilizes Bcl-6 expression. a, Immunoassay of lysates of purified CD44 + CD4 + T cells from pooled OT-II ( n = 8), OT-II OPN-KO ( n = 10) and OT-II OPN-i-KI ( n = 8) mice 7 d post-immunization with OVA in CFA, assessed by immunoprecipitation (IP) with anti-Bcl-6 and immunoblot analysis, as indicated. b, Immunoassay of lysates of 293T cells transfected with plasmids encoding OPN-i and Flag–Bcl-6 wild-type or Flag–Bcl-6 deletion mutants (lanes 1,4,7,10 in ), assessed by IP with anti-Flag and immunoblot analysis, as indicated. Bottom, ratios of precipitated OPN to Bcl-6. c, Immunoblot analysis of lysates of purified CD62L − CD4 + T cells from the indicated OT-II mice 2.5 d post-immunization with OVA in CFA, followed by resting for 2 h, treatment with or without MG132 90 m after incubation with anti-CD3 and anti-ICOS, addition of cycloheximide (CHX) 30 m later, and analysis 3 h after treatment with or without CHX. Right, ratios of Bcl-6 to actin protein. d, Bcl-6 and OPN expression in 293T cells transfected with vectors expressing Flag–Bcl-6 and/or OPN-i, treated with CHX (100 μg/ml) for 10 h. Right, percent of residual Bcl-6 protein relative to that prior to addition of CHX. e–f , Immunoassay of lysates of 293T cells transfected with the indicated plasmids and pre-treated with MG132, assessed by denaturation of lysates, IP with anti-Bcl-6 ( e ) or anti-HA ( f ) and immunoblot analysis as indicated. Bcl-6(Ubn): polyubiquitinated Bcl-6. Increasing amounts of OPN-i plasmids in f. g, Immunoblot analysis of lysates of purified CD62L − CD4 + T cells from indicated OT-II mice 7 d post-immunization with OVA in CFA, treated with (+) or without (−) DUBi for 8 h, probed with anti-Bcl-6 and anti-actin. Bottom, ratios of Bcl-6 to actin. Data represent two ( a–d, g ) and three ( e, f ) independent experiments.

Article Snippet: CD62L − CD4 + T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4 + CD62L + T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining.

Techniques: Expressing, Purification, Immunoprecipitation, Western Blot, Transfection, Incubation