Journal: Frontiers in Immunology

Article Title: Ibrutinib directly reduces CD8+T cell exhaustion independent of BTK

doi: 10.3389/fimmu.2023.1201415

Figure Lengend Snippet: Ibrutinib rejuvenates in vitro exhausted CD8+ T cell functions. (A) Scheme of testing ibrutinib effects on in vitro CTL exhaustion. By repeatedly stimulating the purified OT-І T cells with OVA(257-264) peptide for 5 days, in vitro exhausted CTLs were induced. From day 5, the cells were treated with DMSO or 1μM ibrutinib. On day 8, function and phenotype of the cells were determined. (B) Representative histograms depicting the expression of inhibitory receptors and isotype controls on DMSO or ibrutinib treated exhausted cells on day 8 of the in vitro exhaustion culture. (C) Pooled data showing the median fluorescence intensity (MFI) of the inhibitory receptors expressed on DMSO or ibrutinib treated cells. (D) Bar chart depicting frequency of cells expressing either one, two, three or four of the inhibitory receptors (IRs) PD-1, Lag3, Tim-3 and Tigit. (E) Representative FACS plots illustrating percentage of cytokine-producing CD8+ T cells after OVA(257-264) peptide re-stimulation and intracellular cytokine staining. Exhausted OT-I CD8+ T cells were re-stimulated on day 8 for 6 hours with OVA(257-264) peptide and intracellular cytokines were measured by flow cytometry. (F) Pooled data showing the frequency of cytokine producing CD8+ T cells. (G) Bar graph depicting the frequency of cells producing either one, two or three of the cytokines (IL-2, TNF-α and IFN-γ) simultaneously. Each symbol represents one animal (n=6), 4 independent experiments performed. Lines depict mean ± SE. Between the groups, paired-t test was performed, except for MFI of PD-1, CD160 and Tim3, where the Wilcoxon matched-pairs signed rank test was used “ns” indicates not significant (p>0.05) *P<0.05, **P<0.01.

Article Snippet: For surface staining, cells were first washed with FACS wash (HBSS containing 3% FBS and 0.02% sodium azide) and then incubated at 4°C in the dark for 20 minutes with appropriate concentrations of fluorochrome-conjugated monoclonal antibodies and appropriate isotype controls: anti-CD8a-eFluor 450 (clone 53-6.7, eBioscience), anti-CD160-PE-CF594 (clone CNX46-3, BD Biosciences), anti-Lag3-APC (clone C9B7W, BD Biosciences), anti-CD244-PE (clone 2B4, BD Biosciences; eBio244F4, eBioscience), anti-PD-1-APC-Cy7 (clone 19F.1A12, Biolegend), anti-Tim3-PE-Cy7 (clone RMT3-23, Invitrogen), anti-TIGIT-FITC (clone GIGD7, eBioscience); anti-CD44-BV786 (clone IM7, BD Biosciences).

Techniques: In Vitro, Purification, Expressing, Fluorescence, Staining, Flow Cytometry

Journal: Cancer Communications

Article Title: Enhancement of CAR‐T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins

doi: 10.1002/cac2.12452

Figure Lengend Snippet: Screening of immune checkpoints on CD8 + TILs in cholangiocarcinoma microenvironment. (A) Representative flow plots of immune checkpoints on CD8 + TILs. The proportion of Lag3 + cells in CD8 + TILs of tumor tissue, paratumor tissue, and normal tissue from one patient. (B‐I) Quantitative analysis of immune checkpoints on CD8 + TILs. PD‐1 (B), Tim‐3 (C), Lag3 (D), Tigit (E), BTLA (F), CTLA4 (G), CD244 (H), and CD160 (I) were detected by flow cytometric analysis respectively. The connecting lines indicated that the tissue came from one patient ( n = 15). (J) Statistical analysis of immune checkpoints on CD8 + TILs in cholangiocarcinoma tissue. Experiments were performed independently at least 3 times. Paired two‐tailed student's t‐test was used to compare two non‐independent groups. Experimental data are expressed as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Abbreviations: TIL, tumor‐infiltrating lymphocyte; PD‐1, programmed death‐1; Tim‐3, T‐cell immunoglobulin and mucin domain‐3; Tigit, T cell immunoglobulin and ITIM domain; Lag3, lymphocyte activation gene 3; BTLA, B and T lymphocyte attenuator; CTLA4, cytotoxic T lymphocyte‐associated protein‐4; SEM, standard error of the mean.

Article Snippet: For T‐cell phenotypes in tumor tissues, the following antibodies were used: mouse anti‐human APC‐CD8a (Cat No. 17‐0086‐42, eBioscience, San Diego, CA, USA), PE/CY7‐PD‐1 (Cat No. 561272, BD Biosciences), Brilliant Violet 421‐Tim‐3 (Cat No. 565536, BD Biosciences), PE‐Lag3 (Cat No. 12‐2239‐41, eBioscience), PerCP‐eFluor 710‐Tigit (Cat No. 46‐9500‐42, eBioscience), PE/CY7‐BTLA (Cat No. 344516, Biolegend), PerCP‐eFluor 710‐CTLA4 (Cat No. 46‐1529‐42, eBioscience), Pacific Blue‐CD244 (Cat No. 329524, Biolegend) and PE‐CD160 (Cat No. 12‐1609‐42, eBioscience).

Techniques: Two Tailed Test, Activation Assay

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

Article Title: MicroRNA-139 Expression Is Dispensable for the Generation of Influenza-Specific CD8 + T Cell Responses

doi: 10.4049/jimmunol.2000621

Figure Lengend Snippet: Lack of miR-139 expression does not alter the development of T cell exhaustion. “Effector” and “exhausted” CD8+ T cells were generated in vitro from OT-I or miR-139−/− OT-I mice through either single (Single Stim) or repeated (Repeated Stim) OVA-peptide stimulations and compared with nonstimulated (No Stim) controls. (A) Dot plots of cytokine-producing OT-I cells and (B) dot plot of the frequency of polyfunctional CD8+ T cells 5 d after initial activation in vitro. (C) Dot plots of the frequency or expression level (MFI) of PD-1, LAG3, TIM3, TIGIT, and CD160 in in vitro exhausted WT or miR-139−/− OT-I CD8+ T cells. Representative of four or five mice each from four or five independent experiments. In all figures, error bars represent the SEM.

Article Snippet: Cells were stained with the following fluorochrome-conjugated mAbs: anti-CD8a (clone 53–6.7), anti-CD45.1 (clone A20), anti-CD45.2 (clone 104), anti-Thy1.1 (clone HIS51) (all from eBioscience), anti-CD25 (clone PC61), anti-CD69 (clone H1.2F3), anti-CD44 (clone 1M7), anti-CD62L (clone MEL-14) (all from BD Biosciences), anti-KLRG1 (clone 2F1/KLRG1), anti–IL-7R/CD127 (clone A7R34), anti-PD-1 (clone 29F-IAI2) (all from BioLegend), anti-LAG3 (clone C9B7W), anti-CD160 (clone CNX46–3) (both from BD Biosciences), anti-TIGIT (clone GIGD7; eBioscience), and anti-TIM3 (clone RMT3–23; Invitrogen).

Techniques: Expressing, Generated, In Vitro, Activation Assay

Journal: Immunity & Ageing : I & A

Article Title: Phenotypic and functional alterations of monocyte subsets with aging

doi: 10.1186/s12979-022-00321-9

Figure Lengend Snippet: Percentage of monocyte subsets expressing co-inhibitory molecules decreased in older adults. A . Representative flow cytometry histograms display the expression of the co-inhibitory molecules 2B4, T-cell immunoglobulin domain and mucin domain 3 (TIM-3), CD200R, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), B and T lymphocyte attenuator (BTLA), CD160, programmed death-1 (PD-1), and lymphocyte-activation gene 3 (LAG-3) on monocyte subsets from young adults. B . The percentage of monocyte subsets expressing TIM-3, 2B4, CD200R, TIGIT, and BTLA from young, middle-aged, and older adults (young: 21–40 years, n = 42; middle-aged: 41–60 years, n = 34; older: > 60 years, n = 34). P-values were calculated using the non-parametric Kruskal–Wallis rank test, followed by post hoc analysis

Article Snippet: The gating strategy used is shown in Fig. S . Antibodies used included anti-human CD160-Alexa Fluor 488, CD4-APC-Fire750, CD8-BV510, HLA-DR-Alexa Fluor 700, CD14-APC, PD-1-PE, 2B4-PE-CF594, CD16-BV711, TIM-3-BV650, CD200R-PE, BTLA-BV650, CD45-BV786 (BD Biosciences, San Diego, CA, USA), CX3CR1-BV421, CD3-PerCP-Cy5.5, CD15-PerCP-Cy5.5, CD19-PerCP-Cy5.5, CD29-Alexa Fluor 488, CD62L-BV650, CD11b-BV605, CCR2-PE (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7, and LAG-3-APC (eBioscience, San Diego, CA, USA), along with the corresponding isotype controls.

Techniques: Expressing, Flow Cytometry, Activation Assay