Journal: Nature immunology

Article Title: Phosphoinositide acyl chain saturation drives CD8 + effector T cell signaling and function

doi: 10.1038/s41590-023-01419-y

Figure Lengend Snippet: a, Experimental scheme. WT CD8+ T cells were stimulated (Stim) with anti-CD3, anti-CD28 and IL-2, or cultured with IL-7 (Unstim), for three d. b,c, Relative and absolute lipid intensities, respectively. n = 9 biologically independent samples from three independent experiments; ordinary one-way analysis of variance ANOVA (b) or two-way ANOVA (c) corrected for multiple comparisons (Sidak test). d, Relative intensity (log2 fold change) of saturated PI normalized to unstimulated n = 9 biologically independent samples from three independent experiments; one-sample t-test. e, Percentage PI saturation. n = 9 biologically independent samples from three independent experiments; unpaired two-tailed t-test comparing saturated PIs. f, Total PI concentration (nmol/cell). n = 9 biologically independent samples from three independent experiments; unpaired two-tailed t-test. g, PIPn synthesis and interconversion scheme. h, Total (left) and percentage (right) PIP2 saturation. n = 3 biologically independent samples, representative of two independent experiments; unpaired two-tailed t-test comparing the percentage of saturated PIP2; total polyunsaturated PIP2 = not significant. i, Total (left) and percentage (right) PIP3 saturation. n = 3 biologically independent samples, representative of two independent experiments; unpaired two-tailed t-test comparing the total or percentage saturated PIP3; total polyunsaturated PIP3 = P < 0.001. j, Experimental scheme. OT-I CD45.2+ CD8+ T cells (OT-I) were adoptively transferred into CD45.1+ C57BL/6 mice (1 × 106 cells per mouse). One day later mice were infected intravenously (i.v.) with LmOVA. Four d later, WT CD45.2+/CD8+ T cells were isolated from infected (Lm) or uninfected (CTRL) mice. k–m Total PI concentration (nmol/cell), percentage PI saturation and relative intensity (log2 fold change) of saturated PI normalized to CTRL. n = 3 Ctrl and 12 Lm biologically independent samples; unpaired two-tailed t-test: total PI, percentage saturated PI; one-sample t-test: relative saturated PI. n, Experimental scheme. Sex-matched C57BL/6 mice were injected in the right flank with 1 × 106 B16-F10-OVA cells. Tumor growth was measured up to an average of 7 mm in diameter, then CD8+ T cells were isolated from the spleen (SP) or tumor (TIL). o–q, Total PI concentration (nmol/cell), percentage PI saturation and relative intensity (log2 fold change) of saturated PI normalized to SP. n = 5 biologically independent samples; unpaired two-tailed t-test: total PI, percentage saturated PI; one-sample t-test: relative saturated PI. Error bars show the s.e.m. a.u., arbitrary units.

Article Snippet: OT-I splenocytes were stimulated with plate-bound anti-CD3 (5 μg ml −1 ) (InVivoMab anti-mouse CD3, BioXCell, BE0002), soluble anti-CD28 (0.5 μg ml −1 ; InVivoMab anti-mouse CD28, BioXcell, BE0015) and 100 U ml −1 IL-2 (Peprotech) for 48 h, then cultured for a further 24 h in IL-2 plus vehicle or CDIPTi.

Techniques: Cell Culture, Two Tailed Test, Concentration Assay, Infection, Isolation, Injection

Journal: Nature immunology

Article Title: Phosphoinositide acyl chain saturation drives CD8 + effector T cell signaling and function

doi: 10.1038/s41590-023-01419-y

Figure Lengend Snippet: a, Pie chart showing the number of lipid species measured by LC-QqQ-MS/MS in each of the 18 lipid subclasses. b-c, WT CD8+ T cells were stimulated (Stim) with anti-CD3, anti-CD28 and IL-2 or cultured with IL-7 (unstim) for 3 days. b, Median lipid content per million cells before (raw) and after (normalized) quantile normalization is shown. n = 9 biologically independent samples across 3 independent experiments; unpaired two-tailed t-test. c, Relative total PI level calculated from quantile normalized lipid peak areas. n = 9 biologically independent samples across 3 independent experiments; unpaired two-tailed t-test. d, Experimental schematic. e-f, Volcano plot shows the log2 fold change (e) or total PI (f) between Unstim and Stim conditions using quantile normalized lipid peak areas. n = 3 biologically independent samples; one-way ANOVA or two-way ANOVA corrected for multiple comparisons (Sidak test). g, Relative intensity of saturated PI normalized to Unstim (log2 fold change). n = 3 biologically independent samples; one sample t-test. h, PI saturation percentage. n = 3 biologically independent samples; unpaired two-tailed t-test comparing saturated PI. I, Total PI. n = 3 biologically independent samples; unpaired two-tailed t-test. j, Total PI concentration (nmol/cell). n = 3 biologically independent samples; unpaired two-tailed t-test. k, Saturation of acyl chains in human TE is expressed as a relative proportion of the total PIP2 (left panel) and as a percentage of total PIP2 (right panel). n = 5 biologically independent samples; unpaired two-tailed t-test comparing the total or percentage saturated PIP2. Total polyunsaturated PIP2 was not significantly different. l, Saturation of acyl chains in human TE is expressed as a relative proportion of the total PIP3 (left panel) and as a percentage of PIP3 (right panel). n = 5 biologically independent samples; unpaired two-tailed t-test comparing the total or percentage saturated PIP3. Total polyunsaturated PIP3 was also significantly different (p < 0.001). m, Experimental schematic. n, Percentage CD8+ cells, activation marker HLA-DR+ cells, and proliferation marker Ki67+ cells are shown. MFI of HLA-DR and Ki67 are also shown. o, Relative intensity of saturated PI normalized to HD (log2 fold change). n = 3 HD and n = 3 EBV biologically independent samples; one sample t-test. p, PI saturation. n = 3 HD and n = 3 EBV biologically independent samples; unpaired two-tailed t-test comparing the summed percentage of saturated PI. q, Total PI concentration (nmol/cell). n = 3 HD and n = 3 EBV biologically independent samples; unpaired two-tailed t-test. Error bars show the standard error of the mean. a.u., arbitrary units.

Article Snippet: OT-I splenocytes were stimulated with plate-bound anti-CD3 (5 μg ml −1 ) (InVivoMab anti-mouse CD3, BioXCell, BE0002), soluble anti-CD28 (0.5 μg ml −1 ; InVivoMab anti-mouse CD28, BioXcell, BE0015) and 100 U ml −1 IL-2 (Peprotech) for 48 h, then cultured for a further 24 h in IL-2 plus vehicle or CDIPTi.

Techniques: Tandem Mass Spectroscopy, Cell Culture, Two Tailed Test, Concentration Assay, Activation Assay, Marker

Journal: Nature immunology

Article Title: Phosphoinositide acyl chain saturation drives CD8 + effector T cell signaling and function

doi: 10.1038/s41590-023-01419-y

Figure Lengend Snippet: a–c, WT CD8+ T cells were stimulated with anti-CD3, anti-CD28 and IL-2 for 6 h. Intracellular lipids were analyzed by LC–QqQ–MS/MS or liquid chromatography quadrupole time-of-flight mass spectrometry (LC–QTOF–MS/MS). a, Total PIP2 at indicated time points after activation. n = 5 biologically independent samples; unpaired two-tailed t-test comparing total PIP2. b, Increase in PIP2 species with 0–2 versus ≥3 double bonds between zero h and six h after activation. n = 5 biologically independent samples; unpaired two-tailed t-test. c, Total PI at indicated time points after activation. n = 5 biologically independent samples; unpaired two-tailed t-test comparing total PI. d–h, WT CD8+ T cells were isolated from C57BL/6 mice, then CRISPR–Cas9 technology was utilized to delete CDIPT (CDIPT−) compared to control (CTRL) or ZAP70 deletion (ZAP70−). Cells were stimulated with anti-CD3, anti-CD28 and IL-2 for 48 h, or IL-7 for 48 h (unstimulated control) and T cell activation was assessed. e, CD8+ T cells were gated on Live/Dead-aqua−, then CD44+ and CD62L−. f, Expression of CD25 was quantified after gating on Live/Dead-aqua−CD8+. g, Expression of PD-1 was quantified after gating on Live/Dead-aqua− CD8+. h, Polar metabolites were extracted from supernatants and glucose was measured by LC–MS. Depletion of extracellular glucose across conditions is shown, normalized to cell number. In e–h, n = 4 biologically independent samples; one-way ANOVA corrected for multiple comparisons (Dunnett test). i, Experimental scheme. WT CD8+ T cells were stimulated with anti-CD3, anti-CD28 and IL-2 for 24 h (j), or 48 h (k–n) in the presence or absence of Soy PI (100 μM). j, Total (left) and percentage (right) PI saturation. n = 3 biologically independent samples; one-way ANOVA corrected for multiple comparisons (Tukey test) comparing saturated PI. k,l, Expression of CD25 (k) and CD69 (l) in Live/Dead-IR−, CD8-Brilliant Violet 421+ cells. n = 4 unstimulated and n = 5 stimulated biologically independent samples, representative of three independent experiments; one-way ANOVA corrected for multiple comparisons (Sidak test). m, CD8+ T cells were gated on Live/Dead-aqua−, then CD44+ and CD62L−. n, Intracellular expression of IFN-γ was quantified by flow cytometry. Cells were gated on Live/Dead-IR−, CD8-Brilliant Violet 421+. In m and n, n = 5 biologically independent samples; one-way ANOVA corrected for multiple comparisons (Sidak test). Error bars show the s.e.m.

Article Snippet: OT-I splenocytes were stimulated with plate-bound anti-CD3 (5 μg ml −1 ) (InVivoMab anti-mouse CD3, BioXCell, BE0002), soluble anti-CD28 (0.5 μg ml −1 ; InVivoMab anti-mouse CD28, BioXcell, BE0015) and 100 U ml −1 IL-2 (Peprotech) for 48 h, then cultured for a further 24 h in IL-2 plus vehicle or CDIPTi.

Techniques: Tandem Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry, Activation Assay, Two Tailed Test, Isolation, CRISPR, Expressing, Liquid Chromatography with Mass Spectroscopy, Flow Cytometry

Journal: Nature immunology

Article Title: Phosphoinositide acyl chain saturation drives CD8 + effector T cell signaling and function

doi: 10.1038/s41590-023-01419-y

Figure Lengend Snippet: a, Schematic of PIP2 signaling in Teff cells. b–g, WT CD8+ T cells were stimulated with anti-CD3, anti-CD28 and IL-2 for two d, treated with CTRL or CDIPTi (plus IL-2) for one d and then readouts of downstream signaling were measured. b, Expression of phosphorylated PLCγ1 (left) and activity of PLC enzyme (right). n = 4 biologically independent samples from two independent experiments; unpaired two-tailed t-test. c, Total DAG. n = 4 biologically independent samples representative of three independent experiments; unpaired two-tailed t-test. d, Protein expression of the Raf–MEK1/2–ERK1/2 signaling pathway. e, Relative intensity of p-ERK1/2-PE. n = 3 biologically independent samples; unpaired two-tailed t-test. f, Basal cytoplasmic calcium was calculated using the indo-AM dye bound/unbound ratio. n = 3 biologically independent samples; unpaired two-tailed t-test. g, Relative intensity of Phalloidin-AF647. n = 4 biologically independent samples; unpaired two-tailed t-test. h, Protein expression of the PI3K signaling pathway. i,j, WT CD8+ T cells were stimulated with anti-CD3, anti-CD28 and IL-2 for two d, then a further two d in IL-2. Organelle compartments were enriched and PIPn were extracted and analyzed by LC–QqQ–MS/MS or LC–QTOF–MS/MS. i, PI (upper) or PIP2 (lower) saturation across organelles. n = 4 biologically independent samples. j, Heat map of the relative amount of saturated PIP2 across organelles. n = 4 biologically independent samples; one-way ANOVA corrected for multiple comparisons (Dunnett test) comparing all groups with the lipid rafts. k, WT CD8+ T cells were stimulated with anti-CD3, anti-CD28 and IL-2 for 24 h or 72 h (the final 24 h of which were only with IL-2). Expression of total and phosphorylated PLCγ1, MEK1/2 and ERK1/2 with tubulin as the loading control. n = 4 biological replicates pooled into one lane. l, Lower left, experimental schematic. Membrane-coated beads were generated by mixing liposomes (harboring 16:0–18:1 PI(4)P or 18:0–20:4 PI(4) together with 0.1% Atto647N-DOPE or NBD-DPPE) and silica beads. Purified PLC-PH fused to RFP was added, followed by PIP5K1C immediately before acquisition. Upper microscopy images. Lower right, recruitment of PLC measured by the normalized MFIs of the RFP signal to either of the membranecoated bead populations, segmented using the NBD or Atto647N signal. n = 40–50 individual beads. Error bars show the s.e.m.

Article Snippet: OT-I splenocytes were stimulated with plate-bound anti-CD3 (5 μg ml −1 ) (InVivoMab anti-mouse CD3, BioXCell, BE0002), soluble anti-CD28 (0.5 μg ml −1 ; InVivoMab anti-mouse CD28, BioXcell, BE0015) and 100 U ml −1 IL-2 (Peprotech) for 48 h, then cultured for a further 24 h in IL-2 plus vehicle or CDIPTi.

Techniques: Expressing, Activity Assay, Two Tailed Test, Tandem Mass Spectroscopy, Membrane, Generated, Liposomes, Purification, Microscopy

Journal: Nature immunology

Article Title: Phosphoinositide acyl chain saturation drives CD8 + effector T cell signaling and function

doi: 10.1038/s41590-023-01419-y

Figure Lengend Snippet: a, WT CD8+ T cells were isolated from spleens and lymph nodes of C57BL/6 mice then stimulated with plate-bound anti-CD3 (5 μg/ml), soluble anti-CD28 (0.5 μg/ml) and IL-2 (100 U/ml) for 2 days. Samples for RNA sequencing were harvested every 24 h. Heatmap depicting the gene expression levels of Lclat1 and Mboat7 in n = 3 biologically independent samples. b, WT CD8+ T cells were isolated and activated as in (a), followed by a treatment for 24 h with the Stearoyl-CoA-desaturase inhibitor A939572 (SCDi, 100 nM) in the presence of IL-2. Left panel: Replication Index based on Cell Trace Violet dilution calculated using FlowJo software. Middle and right panel: intracellular expression of IFN-γ. Cells were gated on Live/Dead-IR−, CD8-APC+. n = 3 biologically independent samples; unpaired two-tailed t-test. c, d WT CD8+ T cells were isolated and activated as in (a), followed by culture in IL-2 for 24 h. On d3, cells were either switched to IL-15 (100 U/ml, TM) or maintained in IL-2 until d6. TM were re-activated on d6 by stimulation with plate-bound anti-CD3 (5 μg/ml), soluble anti-CD28 (0.5 μg/ml) and IL-2 (100 U/ml) for 2 days to generate secondary TE. c, Relative amount of PI 36:2 measured every 24 h. n = 3 biologically independent samples; one-way ANOVA corrected for multiple comparisons (Dunnett test) compared all groups to 0 h timepoint. d, Saturation of acyl chains expressed as a proportion of total PIP2 (left panel) and as a percentage of PIP2 (right panel). Statistical analysis of the saturated PI on n = 5 independent biological samples; one-way ANOVA with Dunnett’s multiple comparisons’s test compared all groups to ‘Tn d3’. e, WT CD8+ T cells were differentiated into TM as in (c), with the addition of 100% U-13C-glucose 24 h before lipid analysis at each timepoint. Unstimulated cells were cultured in IL-7 with 100% U-13C-glucose. Fractional contribution of 13C-glucose-derived carbon to PI species was calculated. n = 3 biologically independent samples, two-way ANOVA with correction for multiple comparisons (Sidak’s test) comparing PI species in each group with the relevant 24 h unstimulated PI. Error bars show the standard error of the mean.

Article Snippet: OT-I splenocytes were stimulated with plate-bound anti-CD3 (5 μg ml −1 ) (InVivoMab anti-mouse CD3, BioXCell, BE0002), soluble anti-CD28 (0.5 μg ml −1 ; InVivoMab anti-mouse CD28, BioXcell, BE0015) and 100 U ml −1 IL-2 (Peprotech) for 48 h, then cultured for a further 24 h in IL-2 plus vehicle or CDIPTi.

Techniques: Isolation, RNA Sequencing Assay, Expressing, Software, Two Tailed Test, Cell Culture, Derivative Assay

Journal: Molecular Immunology

Article Title: Death associated protein kinase 2 suppresses T-B interactions and GC formation

doi: 10.1016/j.molimm.2020.10.018

Figure Lengend Snippet: Dapk2 deficiency in T cells leads to increased GC responses. (A) Expression of Dapk2 mRNA increases upon TCR activation. The mRNA level of Dapk2 is analyzed by qPCR and normalized to the level of Hprt, before and after in vitro activation of T cells through crosslinking of CD3 and CD28. Data are mean ± s.e.m. and representative of two independent experiments. (B) Western blot analysis of Dapk2 protein levels in naïve and in vitro activated CD4 + T cells. Data are representative of two independent experiments. (C—E) Adoptive transfer of dsRed OT-2 T cells from WT or Dapk2 knockout mice together MD4 B cells into Tcrb −/− Tcrd −/− recipients. Frequencies of transferred GC B, OT-2 T and Tfh cells in the recipient mice are shown as flow cytometry contour plots (Left) and statistical quantifications (Right). Each symbol represents the mean of one experiment, with at least 3 mice in each group per experiment. Symbols connected are from the same experiment. NS, not significant. *P < 0.05 (paired t -test).

Article Snippet: For in vitro T cell activation, CD4 T cells were stimulated with 8 μg/mL plate-bound anti-CD3 and anti-CD28 antibodies (BioXCell), and cultured in complete RPMI medium containing 10 ng/mL IL-2 (PeproTech) for 5 days before being used in subsequent experiments.

Techniques: Expressing, Activation Assay, In Vitro, Western Blot, Adoptive Transfer Assay, Knock-Out, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: The TYK2 -P1104A Autoimmune Protective Variant Limits Coordinate Signals Required to Generate Specialized T Cell Subsets

doi: 10.3389/fimmu.2019.00044

Figure Lengend Snippet: Healthy subjects expressing the TyK 2 protective variant exhibit decreased proportion of circulating Tfh and switched memory B cells. (A) Gating strategy for T follicular helper (Tfh) cells, defined as CD4 + CD45RA − CXCR5 + PD1 + T cells. Shown are representative dot plots of the Tfh cell frequency in subjects with TYK2 NP / NP ( NP/NP ), TYK2 NP / P ( NP/P ), or TYK2 P / P ( P/P ) (non-protective ( NP ) vs. protective ( P ) alleles of rs34536443; encoding for Alanine-1104 vs. Proline-1104). (B) Quantification of Tfh cell frequency. (C) Gating strategy for switched memory B cells, defined as plasmablast (PB) negative CD3 − CD19 + CD27 + CD10 − IgM − B cells. Shown are representative dot plots of the switched memory B cell frequency in subjects. (D) Quantification of switched memory B cell frequency (Each symbol represents an individual donor) (B,D) ; small horizontal lines indicate the mean (± s.d.). Data from a combined total of n = 19 Tyk2 NP / NP donors, n = 19 TYK2 NP / P donors, and n = 2 TYK2 P / P donors (A,B) ; n = 15 TYK2 NP / NP donors, n = 13 TYK2 NP / P donors, and n = 4 TYK2 P / P donors (C,D) . Statistical analysis indicated from a Mann-Whitney U (B,D) .

Article Snippet: CD4 + T cells were positively isolated (Miltenyi Biotec) and placed into wells that were coated with anti-CD3/CD28 (5 μg/ml; 145-2c11/37.51; BioXcell and UCSF Monoclonal Antibody Core).

Techniques: Expressing, Variant Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: The guanine nucleotide exchange factor Rin-like acts as a gatekeeper for T follicular helper cell differentiation via regulating CD28 signaling

doi: 10.1101/2022.06.23.497284

Figure Lengend Snippet: RNA was isolated from sorted WT and Rinl-KO naïve CD4 + T cells (CD62L + CD44 − ) and subjected to RNA sequencing. (A) Volcano plot depicts the comparison of global gene expression profiles between WT and Rinl-KO naïve CD4 + T cells. The y-axis indicates adjusted p-values (-log10), the x-axis shows the log2 fold change. (B) Diagram shows top dysregulated upstream regulators identified by Ingenuity Pathway Analysis (Qiagen Inc). Regulators are plotted on y-axis and are predicted to be up-(positive z-score, red) or downregulated (negative z-score, blue) in Rinl-KO naïve CD4 + T cells. (C) Summary of CD28 internalization kinetic in naïve and effector CD4 + T cells. Internalization was calculated as percentage (%) of CD28 expression from time point 0 (T=0). (D) Summary of phosphorylation of signaling molecules 2 minutes after αCD3ε or αCD3ε+αCD28 stimulation of CD4 + T cells. (E) Representative histograms of Erk1/2 (pT202/pY204) of WT and Rinl-KO CD4 + T cells activated with immobilized αCD3+αCD28 for 48 hours are shown. Summary is depicted below. (F) Representative histograms of proliferation after activation of naïve CD4 + T cells with low (0,75 μg/mL) and high (3 μg/mL) αCD28 for 3 days. Summary of data is shown alongside. (G) Summary of IL-2 measured from supernatants of cells cultured as in (F). (H) Expression of Cxcr5 was determined by RT-qPCR in naïve CD4 + T cells stimulated as described in (F). (I) Experimental setup. (J) Representative contour plots of Tfh of WT and Rinl-KO mice treated with isotype control or αCD28 (Clone 37.15) one day before immunization. (K) Summary of (J) . Data show a summary of 6-7 (C) , 3-4 (D) , 5 (E) 6 (F , G) , 4 (H) and 4-7 (J , K) mice per group analyzed in 3-4 (C , D, F, G, H) , 5 (E) or 2 (J , K) independent experiments. Data were statistically analyzed using two-way ANOVA using Tukey multiple comparison’s test (C) , paired two-tailed (E) or unpaired two-tailed t-tests (F , K) . For the comparison of the relative geoMFI values between WT (set as 1 for each phospho protein) and Rinl-KO, a one-sample t-test was performed (D) . *p < 0.05, **p < 0.01.

Article Snippet: For the in vivo CD28 study, 100 μg/mL of anti-mouse CD28 (Clone 37,15, BioXcell) or isotype control (isotype poly Syrian hamster IgG, BioXcell) were injected i.p. one day before immunization.

Techniques: Isolation, RNA Sequencing Assay, Expressing, Activation Assay, Cell Culture, Quantitative RT-PCR, Two Tailed Test