Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cholesterol induction in CD8 + T cell exhaustion in colorectal cancer via the regulation of endoplasmic reticulum-mitochondria contact sites

doi: 10.1007/s00262-023-03555-8

Figure Lengend Snippet: High cholesterol promotes CRC by inducing exhaustion in CD8+ T cells. A Workflow of this part. B histogram of cholesterol (Tc), HDL, LDL, Apo(a) and ApoB contents in CRC patients and healthy individuals (ncontrol = 98; nCRC = 217). C IF assay was used to detect the expression of CTLA-4, PD1 and TIM-3 in CD8+ T cells from peripheral blood in CRC patients with healthy serum cholesterol levels and hypercholesterolemia. D comparison of the peripheral blood cholesterol level between CRC mice model with high cholesterol diets and normal cholesterol diets. E, F Pathological morphology and HE staining of colon tissues from CRC mice model of inflammation induced by AOM/DSS. G Expression of CD69, CTLA-4, PD1 and TIM-3 in CD8+ T cells from non-CRC mice with hypercholesterolemia (high CHOL) and CD8+ T cells from non-CRC mice with normal cholesterol level (Normal CHOL). The red box shows the surface of receptor-positive CD8+ T cells. H Histogram of the contents of the cytokines IFN-γ, TNF-α and IL-2 in peripheral blood from mice in the Normal CHOL group and High CHOL group. I–M MC38 cells were used as the control group, Normal CHOL CD8+ T cells were used as the positive control group, and High CHOL CD8+ T cells were used as the experimental group. The CCK8 method (I) was used to detect the proliferation activity of MC38 cells, a scratch test (J, K) was used to detect the migration ability of MC38 cells, flow cytometry L) was used to detect the percentage of apoptosis in MC38 cells, and Transwell assays (M) were used to detect the invasion ability of MC38 cells. * indicating P < 0.05, ** indicating P < 0.01, *** indicating P < 0.001

Article Snippet: Magnetic cell separation (MACS) of CD8 + T cells from peripheral blood Magnetic isolation of CD8 + T cells from peripheral blood specimens was performed using StraightFrom® Whole Blood CD8 MicroBeads and a human and Whole Blood Column Kit (Miltenyi 130–090-878, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Comparison, Staining, Control, Positive Control, Activity Assay, Migration, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cholesterol induction in CD8 + T cell exhaustion in colorectal cancer via the regulation of endoplasmic reticulum-mitochondria contact sites

doi: 10.1007/s00262-023-03555-8

Figure Lengend Snippet: Contact between the endoplasmic reticulum and mitochondria occurs in CRCs at different cholesterol levels. A Workflow of this part. B The expression of CD8+ T cell ERMC proteins (Fis1 and Bap31, MFN2, VAPB and PTPIP51, VDAC1 and IPR3 and GRP75) in the peripheral blood of CRC patients with normal cholesterol and high cholesterol was detected by WB. C–F IF assay was used to detect the expression and location of Fis1 and Bap31, CoX4 and HSP90B1, VAPB and PTPIP51, VDAC1 and IPR3 and GRP75 in CD8+ T cells from CRC patients with normal cholesterol and high cholesterol levels. In the bar chart, * indicates P < 0.05, ** indicates P < 0.01

Article Snippet: Magnetic cell separation (MACS) of CD8 + T cells from peripheral blood Magnetic isolation of CD8 + T cells from peripheral blood specimens was performed using StraightFrom® Whole Blood CD8 MicroBeads and a human and Whole Blood Column Kit (Miltenyi 130–090-878, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cholesterol induction in CD8 + T cell exhaustion in colorectal cancer via the regulation of endoplasmic reticulum-mitochondria contact sites

doi: 10.1007/s00262-023-03555-8

Figure Lengend Snippet: ERS in exhausted CD8+ T cells induced by high cholesterol. A Workflow of this part. B The expression of the CD8+ T cell ERS proteins CHOP and GRP78 in the peripheral blood of CRC patients with normal cholesterol and high cholesterol was detected by WB. C IF assay was used to detect the expression of CHOP and GRP78 in CD8+ T cells from cancerous tissues in CRC patients with normal cholesterol and hypercholesteremia. D Expression of the ERS-related proteins CHOP and GPR78 in mice from each group detected by WB. E Endoplasmic reticulum morphology of different groups of CD8+ T cells observed by transmission electron microscopy. In the normal CHOL group, the rough endoplasmic reticulum distribution was also reduced. In the High CHOL group (CD8+ T cells from mice in the High cholesterol group), the endoplasmic reticulum was rare. In the MC-38/CD8+ T-WT group (MC-38 cells were cocultured with CD8+ T cells from wild-type mice), endoplasmic reticulum disintegration was observed in some parts. In the MC-38/CD8+ T-high CHOL group (coculture of MC-38 cells with CD8 + T cells from mice in the high cholesterol group), the ER was disintegrated. In the CD8+ T-4-PBA group (mice CD8+ T cells treated with the ERS inhibitor 4-PBA), ER structures were less common in the cytoplasm. In the MC-38/CD8+ T-4-PBA group (intervention with the ERS inhibitor 4-PBA in the coculture system of MC-38 cells and mice CD8+T cells), the arrow shows ERS disintegration. F Histogram and flow chart of PD1, TIM-3, CTLA-4 and CD69 expression in spleen T cells from mice in the 3 groups. G Cell proliferation was detected by the CCK-8 method, and differences were observed among the 3 groups of tumor cells (P < 0.05). H Cell invasion ability detected by the Transwell method. I Cell migration detected by a scratch test. J apoptosis was detected by Annexin-V APC/7-AAD double staining. In the bar chart, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001

Article Snippet: Magnetic cell separation (MACS) of CD8 + T cells from peripheral blood Magnetic isolation of CD8 + T cells from peripheral blood specimens was performed using StraightFrom® Whole Blood CD8 MicroBeads and a human and Whole Blood Column Kit (Miltenyi 130–090-878, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Expressing, Transmission Assay, Electron Microscopy, CCK-8 Assay, Migration, Double Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cholesterol induction in CD8 + T cell exhaustion in colorectal cancer via the regulation of endoplasmic reticulum-mitochondria contact sites

doi: 10.1007/s00262-023-03555-8

Figure Lengend Snippet: Exhaustion of CD8+ T cells induced by high cholesterol showed structural and functional changes in ERMCs. A Workflow of this part. B Confocal immunofluorescence microscopy of mitochondria and ER of CD8+ T cells in the normal cholesterol group, high cholesterol group, normal CHOL CRC group, and high CHOL CRC group. Among these groups, the nucleus was blue, mitochondrial probe was green, and the ER probe was red. The higher the yellow overlap in the merged diagram is, the greater the colocalization of mitochondria and endoplasmic reticulum. C The expression of the mitochondrial fusion protein MFN2 in CD8+ T cells from the 4 groups was detected by Western blot. D–F Co-immunoprecipitation was performed to clarify the interaction between Fis1 and Bap31, VAPB and PTPIP51, and VDAC1 and IPR3 and GRP75 proteins of ERMCs in CD8+ T cells. Input refers to the protein content in cells, and IP refers to the protein content measured by the antigen antibody response. G, Immunofluorescence was performed to clarify the expression and location of MFN2 and CoX4 and HSP90B1, VAPB and PTPIP51, and VDAC1 and IPR3 and GRP75 in CD8+ T cells. The nucleus (blue) and other colors correspond to the probe colors of each molecule. The more orange parts in the combined figure, the more molecules are located in the cell

Article Snippet: Magnetic cell separation (MACS) of CD8 + T cells from peripheral blood Magnetic isolation of CD8 + T cells from peripheral blood specimens was performed using StraightFrom® Whole Blood CD8 MicroBeads and a human and Whole Blood Column Kit (Miltenyi 130–090-878, Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Functional Assay, Immunofluorescence, Microscopy, Expressing, Western Blot, Immunoprecipitation

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 1. CD40L expression on SV40 TAg–specific CD8+ T cells during a protective immune response. (A) Splenocytes of WT mice challenged with 16.113 TAg+ cancer cells were stimulated with peptide IV at indicated time points. d, day. The dot plots show the intracellular IFN-γ and CD40L staining of CD3+CD8+CD4−-gated lymphocytes from one representative mouse (n = 4 mice). (B) The diagram summarizes the frequencies of CD40L+IFN-γ+ and CD40L−IFN-γ+CD8+ T cells and (C) the frequency of IL-2– producing cells among CD40L+ and CD40L− tumor-specific CD8+ T cells at the different time points (both means ± SD).

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 2. Prevention of tumor outgrowth is dependent on CD40L expression on CD8+ T cells. (A) RAG1−/− mice were injected subcutaneously with 1 × 106 9.27 TAg+ cancer cells and treated in parallel with intravenously injected CD8+ T cells from WT or CD40L−/− mice and/or with WT CD4+ T cells. The means ± SD of the tumor size of four or five mice per group are shown from one representative of five experiments. (B) The tumor sizes of individual mice in different groups are shown at day 26. (C) Sum- mary of the tumor formation data obtained from five independent experiments. Tumor formation was defined as an established tumor when its volume reached >500 mm3. Statistical analysis: Analysis of variance (ANOVA) with Bonferroni multiple comparisons posttest: *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Injection

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 3. Impaired tumor rejection in nonlymphopenic CD8+ T cell–specific CD40L KO mice. (A) Strategy for the generation of CD40Lfl/fl mice. UTR, untranslated region; FRT, flippase recognition target. (B) E8I-Cre × CD40Lfl/fl, E8I-Cre, and CD40L fl/fl mice as WT control were injected subcutaneously with 1 × 106 9.27 TAg+ cancer cells. Sum- mary of the tumor formation data obtained from three individual experiments, each with 5 to 10 mice per group. Tumor was defined as established when its volume reached >500 mm3. (C and D) WT and E8I-Cre × CD40Lfl/fl mice were subcutaneously injected with 1 × 106 9.27 TAg+ cancer cells, and 7 days later, splenocytes and cells from the draining lymph nodes (dLNs) were isolated and stimulated with peptide IV. (C) The dot plots show the intracellular IFN-γ and CD40L staining of CD3+CD8+CD4−- gated splenocytes from one representative mouse of five mice. (D) The diagram summarizes the frequencies of TAg-specific IFN-γ+CD8+ T cells measured among spleno- cytes and lymph node cells. Statistical analysis: Log-rank test: **P < 0.01.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Control, Injection, Isolation, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 5. CD8+ T cell–mediated CD40 signaling in cancer cells prevents tumor formation. (A) Cell surface expression of CD40 was analyzed after culturing TRAMP-C1 and CD40tg TRAMP-C1 cells with or without TGFβ for 24 hours. (B) Both TRAMP-C1 cancer cell lines were stimulated for 24 hours with multimeric CD40L. Thereafter, cas- pase-8 activity was determined with the fluorescence marker FITC-IETD-FMK. The representative dot plots show the gating of 4′,6-diamidino-2-phenylindole (DAPI) and FITC-IETD-FMK–stained cancer cells after triggering CD40. The diagrams summarize the frequencies of active caspase-8+ cancer cells. (C and D) E8I-Cre × CD40Lfl/fl and E8I-Cre as WT control mice were injected subcutaneously with 5 × 106 TRAMP-C1 (C) or CD40tg TRAMP-C1 (D) cancer cells. In the diagrams [(C) and (D)], the data from one of two representative experiments are shown. Per group, five or six mice were challenged. Tumor was defined as established when its volume reached >500 mm3. Statis- tical analysis: (B) Mann-Whitney U test: **P < 0.01 and [(C) and (D)] log-rank test: *P < 0.05 and **P < 0.01.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Activity Assay, Fluorescence, Marker, Staining, Control, Injection, MANN-WHITNEY

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 6. CD40L+CD8+ T cells mediate cell death in human CD40+ carcinoma cell lines by caspase-8 activation. (A) Histograms represent the CD40 expression on EJ138 or A704 transfected with nontargeting single guide RNA (black, sgNon) or cells depleted for CD40 by CRISPR-Cas9 and sgRNA against human CD40 (red). Gray-filled his- tograms show isotype staining. (B) sgNon (black) or caspase-8 sgRNA–treated (red) EJ138 and A704 cells were treated with multimeric CD40L for 24 hours, and caspase-8 activation was monitored by FITC-IETD-FMK staining. Gray-filled histograms represent the basal caspase-8 activation of unstimulated WT cells. (C and D) sgNon, CD40 sgRNA-, or caspase-8 (Casp8) sgRNA–treated variants of EJ138 (C) or A704 (D) were cocultivated with CD40L-enriched CD8+ T cells for 24 hours, and apoptosis was de- tected by annexin V and DAPI staining. Shown are representative dot plots with frequencies of technical triplicates (left) and bar graphs summarizing the data from ex- periments with eight different T cell donors. Statistical analysis: [(C) and (D)] Repeated measures ANOVA, followed by Dunnett’s test: **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Activation Assay, Expressing, Transfection, CRISPR, Staining

Journal: Science advances

Article Title: CD8 + T cell-derived CD40L mediates noncanonical cytotoxicity in CD40-expressing cancer cells.

doi: 10.1126/sciadv.adr9331

Figure Lengend Snippet: Fig. 7. Resistance pattern for CD40-mediated cell death and correlations between CD8 and CD40L in different RCC cohorts. (A) In the histograms, the dark gray areas display the CD40 expression, and the light gray areas display the corresponding isotype control on eight different RCC lines. The included numbers represent the mean fluorescence intensity fold change between CD40 and isotype control staining. (B) Percentages of CD40L-induced lysis per RCC line after stimulation for 48 hours are plotted. Representative lysis values after backgorund substraction of each cell line of at least three individual experiments are depicted. (C) The heatmap represents the top 10 differentially expressed genes between CD40-resistant and CD40-sensitive RCC cell lines. (D) In the Kaplan-Meier survival plot, the resistance score of low and high groups of the TCGA-KIRC patient cohort is compared with each other. (E) Partial correlation networks of different patient groups are displayed, and the numbers are the partial correlation coefficients. (F) In the Kaplan-Meier survival plots, the resistance score of low and high groups of the cohorts is compared with all patients treated within the checkmate-009, checkmate-010, and checkmate-025 studies (left) or divided into two arms of the checkmate-025 study (middle nivolumab arm and right everolimus arm). Statistical analysis: (C) Described in Materials and Methods; [(D) and (F)] log-rank test.

Article Snippet: The following mice were obtained from the Jackson Laboratory and bred and housed under specific pathogen–free conditions at the institution’s animal facility (Charité): CD40L−/− (RRID: IMSR_JAX:002428), CD4−/− (RRID: IMSR_JAX:002663), CD8−/− (RRID: IMSR_JAX:002665), CD45.1 (RRID: IMSR_JAX:002014), and CD40−/− (RRID: IMSR_ JAX:002928).

Techniques: Expressing, Control, Fluorescence, Staining, Lysis

Journal: Oncoimmunology

Article Title: Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer

doi: 10.1080/2162402X.2022.2046931

Figure Lengend Snippet: Ex vivo expression of NKG2A is highest on CD8 αβ T cells infiltrating colorectal tumors, regardless of MSS/MSI status . ( a ) Distribution of viable NKG2A + lymphoid cells among αβ T cells (CD3 + TCRαβ + cells), γδ T cells (CD3 + TCRγδ + cells), and NK cells (CD3 − CD56 + ) in 20 colorectal tumors; Friedman’s test, followed by Dunn’s multiple comparisons test. ( b ) Representative density plots of NKG2A expression on CD3 + CD8 + T cells in healthy tissues (1.1 and 6.6%, top) and tumor tissues (16.7 and 9.4%, bottom), respectively from C161 and C170 patients. ( c ) Paired frequencies of NKG2A + cells among CD8 T cells in paired tumor and normal tissues (n = 29); Wilcoxon paired t test. ( d ) Frequencies of NKG2A + cells among CD8 + TILs according to MSS/MSI status (n = 35); Mann–Whitney test.

Article Snippet: First, CD8 + TILs were collected by magnetic cell sorting using the REAlease CD8 MicroBead kit by positive selection (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Ex Vivo, Expressing, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer

doi: 10.1080/2162402X.2022.2046931

Figure Lengend Snippet: NKG2A + CD8 + TILs are tissue-resident cells harboring terminally exhausted features . ( a ) Proportion of NKG2A + (dark blue squares) and NKG2A − (light blue circles) CD8 + TILs from 9 colorectal tumors for each T-cell differentiation stage, defined as follows: Naive (CCR7 + CD45RO − CD95 − ), Short-lived effector cell (SLEC) (CCR7 − KLRG1 + CD45RO + CD95 − ), Effector (CCR7 − KLRG1 − CD45RO + CD95 − ), T SCM (CCR7 + CD45RO − CD95 + ), T CM (CCR7 + CD45RO + CD95 + ), T EM (CCR7 − CD45RO + CD95 + ) and T EMRA (CCR7 − CD45RO − CD95 + ). ( b ) Frequencies of CD103 + cells among NKG2A + (dark blue squares) and NKG2A − (light blue circles) CD8 + TILs from 7 colorectal tumors; Wilcoxon paired t test. ( c ) Proportion of NKG2A + (dark blue squares) and NKG2A − (light blue circles) CD8 + TILs from 7 colorectal tumors for each T-cell exhaustion stage, defined as follows: Tex prog1 (TCF1 + CD69 + ), Tex prog2 (TCF1 + CD69 − ), Tex int (TCF1 − CD69 − ) and Tex term (TCF1 − CD69 + ); Wilcoxon paired t test. The percentage of CD103 + cells in the different subgroups is indicated below each histogram.

Article Snippet: First, CD8 + TILs were collected by magnetic cell sorting using the REAlease CD8 MicroBead kit by positive selection (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Cell Differentiation

Journal: Oncoimmunology

Article Title: Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer

doi: 10.1080/2162402X.2022.2046931

Figure Lengend Snippet: NKG2A + CD8 + TILs co-express other ICs at different levels highlighting two distinct subsets . ( a ) Representative histograms showing the expression of PD-1, Tim-3, TIGIT, or Lag3 on NKG2A − (left panels) and on NKG2A + (right panels) CD8 + TILs from C169 patient. Results were expressed as positive cells and Relative Fluorescence Intensity (RFI) defined as the ratio of specific fluorescence (median fluorescence of cells incubated with specific Abs) over non-specific fluorescence (median fluorescence of cells incubated with isotypic controls). ( b ) Paired frequencies (n = 35) of NKG2A − (light blue) and NKG2A + (dark blue) CD8 + TILs expressing PD-1, Tim-3, TIGIT, or Lag3; Wilcoxon paired t test. ( c ) Pie chart analysis of the co-expression of the 4 ICs on NKG2A − and NKG2A + CD8 + TILs. ( d ) Density tSNE plots from FlowJo software on pre-gated viable CD3 + CD8 + NKG2A + TILs after concatenation of all analyzed tissue samples (n = 9). The two clusters identified on the basis of the differential expression of NKG2A are encircled in blue (higher expression) and orange (lower expression). The expression level of the 5 ICs for each cluster is represented below by histograms.

Article Snippet: First, CD8 + TILs were collected by magnetic cell sorting using the REAlease CD8 MicroBead kit by positive selection (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing, Fluorescence, Incubation, Software, Quantitative Proteomics

Journal: Oncoimmunology

Article Title: Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer

doi: 10.1080/2162402X.2022.2046931

Figure Lengend Snippet: Comparative transcriptomic analysis of NKG2A + and NKG2A − CD8 + TILs reveals downregulation of many genes involved in cell proliferation in NKG2A + cells . Volcano plots of differentially expressed mRNAs between NKG2A − and NKG2A + cells unstimulated ( a ) and activated ( c ) (n = 4 pairs), using the EdgeR algorithm. Heatmaps showing the expression scale of up- and down-regulated genes identified by one (light gray), two (dark gray) or three (black) algorithms (DEseq2, EdgeR and/or limma-voom) in NKG2A + cells unstimulated ( b ) and activated ( d ) (n = 4 pairs). Each column represents the average of two technical replicates.

Article Snippet: First, CD8 + TILs were collected by magnetic cell sorting using the REAlease CD8 MicroBead kit by positive selection (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Oncoimmunology

Article Title: Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer

doi: 10.1080/2162402X.2022.2046931

Figure Lengend Snippet: NKG2A + CD8 + TILs have better functional capacity than NKG2A − CD8 + TILs despite reduced proliferative potential . ( a ) Representative histograms of proliferation (CFSE assay) of NKG2A − (up) and NKG2A + (down) CD8 + TILs from C10 patient after a 5-day culture in the presence of OKT3 mAb (from 0 to 200 ng/mL). ( b ) Proportion of proliferative NKG2A − (dashed light blue lines) and NKG2A + (solid blue lines) CD8 + TILs in 4 patients, after a 5-day culture in the presence of OKT3 mAb (from 0 to 200 ng/mL). ( c ) Representative histograms of the percentage of NKG2A − (up) or NKG2A + (down) CD8 + TILs from C81 patient producing IFN-γ, TNF-α, or IL-2 after a 5-hour stimulation (OKT3 mAb, 400 ng/mL) in the presence of brefeldin A (10 µg/mL) and expressing the surface degranulation marker CD107a after 3 hours in the presence of OKT3 mAb (400 ng/mL). ( d ) Percentage of NKG2A − (light blue) or NKG2A + (dark blue) CD8 + TILs (n = 4) producing IFN-γ, TNF-α, IL-2 or CD107a in the same experimental conditions as ( c ).

Article Snippet: First, CD8 + TILs were collected by magnetic cell sorting using the REAlease CD8 MicroBead kit by positive selection (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Functional Assay, CFSE Assay, Expressing, Marker

Journal: Oncoimmunology

Article Title: Targeting NKG2A to boost anti-tumor CD8 T-cell responses in human colorectal cancer

doi: 10.1080/2162402X.2022.2046931

Figure Lengend Snippet: Inhibitory receptor CD94/NKG2A engagement induces impaired IFN-γ secretion in polyclonal NKG2A + CD8 + TILs . ( a ) Histograms showing the purity of the tested NKG2A − and NKG2A + CD8 + TIL populations. ( b ) Percentages of inhibition of IFN-γ secretion by NKG2A − and NKG2A + CD8 + TILs from C65 (purple triangles) and C178 (blue circles) patients in the presence of soluble OKT3 mAb (500 ng/mL) and coated isotypic control (light gray) or agonist anti-CD94 mAb (dark gray) (1 µg/mL). These percentages, calculated relative to the activated condition without antibody, were obtained from 3 independent experiments with 3 replicates (n = 9) for each patient; Wilcoxon paired t test. ( c ) Graphs showing the frequencies of reactive IFN-γ + cells among NKG2A − and NKG2A + CD8 + TIL populations after 5-hour coculture with the HCT116 cell line in the presence of brefeldin A (10 µg/mL).

Article Snippet: First, CD8 + TILs were collected by magnetic cell sorting using the REAlease CD8 MicroBead kit by positive selection (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Inhibition, Control

Journal: PloS one

Article Title: Cross-protective peptide vaccine against influenza A viruses developed in HLA-A*2402 human immunity model.

doi: 10.1371/journal.pone.0024626

Figure Lengend Snippet: Figure 4. An accumulation of murine CD3+ and CD8+ cells around the bronchioles in intranasally immunized mice. A24Tg mice were immunized three times at 7 to 9 days intervals i.n.(A,C,D,E) or s.c.(B) with PA130–138, PB1430–438 and PB2549–557 peptides in the presence of CpG-ODN (B,C,D), Tyrosinase206–214 plus CpG-ODN (E) or CpG-ODN plus empty-liposome solution (A). Lungs were harvested at day 7 after the final immunization, embedded in O.C.T. compound, frozen in dry ice-2-propanol. Ten mm thick frozen sections were prepared. The sections were post- fixed in acetone:ethanol (1:1) solution and blocked endogenous avidin and biotin activity, then stained with anti-mouse CD3 (A,B,C) or anti-mouse CD8a (D,E). doi:10.1371/journal.pone.0024626.g004

Article Snippet: The sections were stained with biotinylated hamster anti-mouse CD3 (eBioscience, San Diego, CA) or biotinylated rat anti-mouse CD8a (R&D Systems ) antibodies.

Techniques: Avidin-Biotin Assay, Activity Assay, Staining

Journal: JCI insight

Article Title: Collectin-11 promotes cancer cell proliferation and tumor growth.

doi: 10.1172/jci.insight.159452

Figure Lengend Snippet: Figure 3. Colec11–/– mice exhibit less immunosuppressive TME. Tumors excised from Colec11+/+ (WT) or Colec11–/– (KO) mice (d14) were used for analyzing TME. (A–C) Tumor infiltrates analyzed by flow cytometry. (A) CD45+ cells. (B) Subsets of tumor-infiltrating leukocytes analyzed by flow cytometry. Data were analyzed by unpaired t test (n = 18 mice per group, pooled from 4 experiments). Each dot represents an individual mouse. (C) A bar chat representing proportion of subsets in CD45+ cells shown in B. (D) Representative microscopy images of immunochemical staining for CD11b (green)/CD3 (red)/DAPI (blue) and F4/80 (green)/CD3 (red)/DAPI (blue). Scale bar: 50 μm. (E) Representative microscopy images of immunochemical staining for CD8 (red)/DAPI (blue) in tumor edge and core areas. Scale bar: 50 μm. (F). qPCR analysis in tumor tissues. Data were analyzed by unpaired t test (n = 8 mice per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following antibodies were used in immunochemical staining: monoclonal rat anti–mouse CD45 (103120), CD11b (101202), and F4/80 (123102) (all from BioLegend); rat anti–mouse CD31(557355, BD Biosciences); rabbit anti-mouse CD3 (ab237721), CD8α (ab217344), VWF (ab6994), and rabbit anti– human COLEC11 (ab238585) (all from Abcam); rabbit anti–mouse H2-Ab1 (A18658, ABclonal); rat anti– mouse CD68 (FA11) (Bio-Rad); goat anti–mouse CD206(AF2535) (from R&D systems); rabbit anti–mouse Ki67 (9129, Cell Signaling Technology); Alexa Fluor 488 goat anti–rat IgG (catalog 405418), Alexa Fluor 555 goat anti–rat IgG (catalog 405420), and Alexa Fluor 647 donkey anti–rabbit IgG (catalog 406414) (all from BioLegend); and Alexa Fluor 488 goat anti–rabbit IgG (catalog 4412) and Alexa Flour 594 goat anti– rabbit IgG (catalog 8889) (both from Cell Signaling Technology).

Techniques: Flow Cytometry, Microscopy, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Scheme 1. The schematic diagram demonstrates the ApoVs interact with CD8+ T cells via membrane fusion, triggering cascade reactions including calcium overload, mitochondrial dysfunction, BAX translocation, and eventual apoptosis in CD8+ T cells.

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Membrane, Translocation Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 1. MSCs-ApoVs Treatment Attenuated CD8+ T Cells-mediated Contact Hypersensitivity. A) Schematic illustration of contact hypersensitivity experimental design. B) Representative phenotype of ears captured by dermoscopy. C) Hematoxylin-eosin (H&E) staining of ear samples collected 24 h after the challenge. The lower panel magnifies the boxed area in the top panel. Scale bar: 150 μm for the upper panel and 50 μm for the lower panel. Black arrowheads indicate dilated capillaries. D) Ear thickness was measured 24 h after the elicitation in three groups (n = 15). E) Volcano plots

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 2. ApoVs-afforded Anti-hypersensitivity Effects by Promoting the Apoptosis of CD8+ T Cells. A) Schemes of the adoptive transfer experimental design. CD8+ T cells from the draining lymph nodes of oxazolone-sensitized WT mice were isolated and cultured with or without ApoVs. Naïve WT mice then received adoptive transfer of these CD8+ T cells, followed by treatment on the ears of recipient mice with OXA 2 h post-transfer. B) Representative ear lesions visualized by dermoscopy. C) H&E staining of ear samples collected 24 h after the challenge. The lower panel magnifies the boxed area in the top panel. Scale bar: 150 μm for the upper panel and 50 μm for the lower panel. Black arrowheads indicate dilated capillaries. D) Ear thickness was measured 24 h after the elicitation in groups receiving intravenous infusion of CD8+ T cells treated with PBS or ApoVs (n = 10). E) Bubble diagram displayed the top 10 KEGG pathways enriched terms of upregulated DEGs in ApoVs-treated CD8+ T cells compared to PBS-treated CD8+ T cells (n = 3). F) The bar chart showed the top 12 Reactome enrichment pathways of upregulated DEGs in ApoVs-treated CD8+ T cells compared to PBS-treated CD8+

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Adoptive Transfer Assay, Isolation, Cell Culture, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 3. ApoVs Induced Apoptosis in CD8+ T cells via Affecting Mitochondrial Morphology and Function. A) Proteomic analysis of ApoVs comparing with EVs showed that GO top 10 enrichment terms of upregulated DEPs, categorized into “Molecular Function” (n = 3). B) The bar chart showed the top 10 Reactome enrichment pathways of upregulated DEPs in ApoVs, compared to EVs (n = 3). C) Doubling the resolution of structured illumination microscopy (SIM2) showed the fragmentation of mitochondrial morphology in CD8+ T cells after ApoVs treatment. Scale bar: 2 μm. D-E) The mean perimeter and mean area of mitochondria in ApoVs treated CD8+ T cells significantly decreased (n = 40). F) Transmission electron microscopy images revealed the mitochondrial morphology. The lower panel magnifies the boxed area in the top panel. Scale bar: 1 μm for the upper panel and 0.5 μm for the lower panel. G) SIM2 exhibited the mitochondrial permeability increased in ApoVs treated CD8+ T group, represented by decreasing of relative fluorescence intensity of Calcein AM (n = 5). Scale bar: 2 μm. H) Flow cytometry analysis exhibited the relative fluorescence intensity of Calcein AM (n = 3). I) Mitochondrial membrane potential (∆Ψm) was analyzed by the relative ration of JC-1 aggregates (OD = 525) and monomer (OD = 490) (n = 5). J) Relative mitochondrial ROS level of two groups (n = 5). ** p < 0.01, ***p < 0.001.

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Microscopy, Transmission Assay, Electron Microscopy, Permeability, Flow Cytometry, Membrane

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 4. ApoVs Evoked Calcium Influx through Membrane Fusion with CD8+ T Cells. A) SIM2 showed the ApoVs (red) fused with the membrane of CD8+ T cells (green) in a time manner. Scale bar: 2 μm. B) Relative fluorescence intensity of PKH26 (ApoVs) enhanced after 6 h in CD8+ T cells treated with ApoVs (n = 3). C) SEM showed a sequential observation of ApoVs contact with CD8+ T cells. D) Cytosolic Ca2+ levels in CD8+ T cells after being treated with ApoVs or PBS in 6 min (n = 5). E) Quantification of the mean maximal [Ca2+] rises (Δ[Ca2+]) in CD8+ T cells when treated with ApoVs or

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Membrane

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Apoptotic Vesicles Derived from Mesenchymal Stem Cells Ameliorate Hypersensitivity Responses via Inducing CD8 + T Cells Apoptosis with Calcium Overload and Mitochondrial Dysfunction.

doi: 10.1002/advs.202407446

Figure Lengend Snippet: Figure 5. Harnessing Calcium Influx in CD8+ T Cells Weaken the Efficacy of ApoVs. A) Cytosolic Ca2+ levels and the mean maximal [Ca2+] rises (Δ[Ca2+]) in CD8+ T cells when stimulated with PBS, ApoVs, and ApoVs pretreatment with 1 μM verapamil groups in 6 min (n = 5). B) Quantification of the mean maximal [Ca2+] rises (Δ[Ca2+]) in CD8+ T cells after being treated with PBS, ApoVs, and ApoVs pretreatment with verapamil groups in 6 min (n = 60). C) SIM2 showed the Calcein AM expression in CD8+ T cells treated with PBS, ApoVs, and ApoVs pretreatment with verapamil respectively. Scale bar: 5 μm. D) Quantification of relative fluorescence intensity of Calcein AM in three groups (n = 5). E) H&E staining of ear samples collected 24 h after the challenge. Scale bar: 150 μm. Black arrowheads indicate dilated capillaries. F) Ear thickness was measured 24 h after the elicitation in groups of intravenous infusion of CD8+ T cells (n = 5). G) SIM2 showed the co-localization of BAX (red) and Mitotracker (green) in three groups respectively. Scale bar: 2 μm. Boxed scale bar: 0.5 μm. H) Quantification of percentage of BAX and Mitotracker co-localized Area (n = 5). I) Western blot displayed the expression level of BAX in isolated mitochondria from CD8+ T cells in three groups. J) Western blot displayed the expression level of classical apoptosis protein cleaved-Caspase 9 and cleaved-Caspase 3 in three groups. * p < 0.05, ** p < 0.01, ***p < 0.001.

Article Snippet: The antibodies used were: anti-CD31 antibody (FAB3628G, R&D Systems, USA; diluted 1:100), anti-TGF-β antibody (3711S, Cell Signaling Technology, USA, diluted 1:200), anti-TNF-α antibody (11948s, Cell Signaling Technology, USA, diluted 1:200), mouse CD8 alpha Alexa Fluor 488 MAb (FAB116G-100, R&D system, USA, diluted 1:200), anti-Cav1.2 antibody (ACC-003, Alomone, Israel, diluted 1:200), anti-Cavβ1 antibody (PA5109285, Invitrogen, USA, diluted 1:200), anti-BAX antibody(60267-1-Ig, Proteitech, China, diluted 1:200), Alexa Fluor 488 Donkey anti-Mouse secondary antibody (A21202, Invitrogen, USA, diluted 1:200), Alexa Fluor 594 Donkey anti-Mouse secondary antibody (A21203, Invitrogen, USA, diluted 1:200), Alexa Fluor 488 Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200) and Alexa Fluor 594 Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200).

Techniques: Expressing, Staining, Western Blot, Isolation