Journal: Frontiers in Immunology

Article Title: Response gene to complement 32 promotes tumorigenesis by mediating DNA damage repair and inhibits CD8+ T cells infiltration in diffuse large B-cell lymphoma

doi: 10.3389/fimmu.2025.1591615

Figure Lengend Snippet: RGC32 knockdown promoted DNA damage and CD8+ T cells. infiltration in DLBCL TME. (A, B) Western blot was conducted to assess the protein levels of phosphorylated (p) ATM/ATR/CHK1, p-H2AX, IRF1 and PD-L1 in cells transfected with shControl and shRGC32. (C, D) In vivo , IHC and flow cytometry showed knockdown of RGC32 increased CD8 expression. (E) IHC images represented the association between expressions of CD8 and RGC32 in DLBCL tissues. Bar = 5μm.

Article Snippet: Cell surface CD8a and CD3 of TILs was stained with CD8a Flow antibody (Proteintech, PE-65069, 0.3μl) and CD3 Flow antibody (Proteintech, APC-65077, 0.5μl), and tested by FACS- 240 Navios Flow Cytometer (Beckman Coulter Inc. USA).

Techniques: Knockdown, Western Blot, Transfection, In Vivo, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Response gene to complement 32 promotes tumorigenesis by mediating DNA damage repair and inhibits CD8+ T cells infiltration in diffuse large B-cell lymphoma

doi: 10.3389/fimmu.2025.1591615

Figure Lengend Snippet: Schematic diagram of pathways regulated by RGC32 in DLBCL. RGC32 promotes DNA damage repair in DLBCL cells by activating the ATM/ATR/CHK1 pathway. The reduction of DNA damage leads to a decrease in tumor neoantigen load, and the ATM/ATR/CHK1 pathway can activate IRF1 to promote PD-L1 expression. Both of these aspects could inhibit the infiltration of CD8+ T lymphocytes in the tumor microenvironment, thereby promoting the occurrence and development of DLBCL.

Article Snippet: Cell surface CD8a and CD3 of TILs was stained with CD8a Flow antibody (Proteintech, PE-65069, 0.3μl) and CD3 Flow antibody (Proteintech, APC-65077, 0.5μl), and tested by FACS- 240 Navios Flow Cytometer (Beckman Coulter Inc. USA).

Techniques: Expressing

Journal: JCI Insight

Article Title: Combined single-cell transcriptome and immune repertoire analysis reveals hepatic and renal immune injury by heat stroke

doi: 10.1172/jci.insight.189825

Figure Lengend Snippet: ( A ) The subclustering of CD8 + T cells identified 6 cell subclusters. ( B ) Dot plot illustrating the scaled expression of representative marker genes across the 6 CD8 + T cell subclusters. ( C ) Pie diagrams displaying the proportion of sample group contributions per annotated CD8 + T cell subclusters. ( D ) Relative contribution of the 6 CD8 + cell subclusters in different sample groups. P values were obtained using the 1-way Kruskal-Wallis test with post hoc Dunn’s test. ( E ) Frequencies of HS-associated CD8 + T cells as determined by flow cytometry. P values were obtained using the 1-way Kruskal-Wallis test with post hoc Dunn’s test. ( F ) Dot plot showing the expression of cytotoxic effector features in CD8 + T cell subtypes derived from the HC, HE, and HS groups. ( G ) Bar plot showing the percentage distribution of TCR clonality types among CD8 + T cell subsets. ( H ) Heatmap illustrating the overlap of TCR clonality types among CD8 + T cell subsets. ( I ) Schematic showing the differentiation trajectory among each CD8 + T cell subset. ( J ) Heatmap displaying dynamic changes in gene expression along the pseudotime of CD8 + T cell differentiation trajectory.

Article Snippet: The following antibodies were used: anti-human CD4 (APC/cyanine, 7317417, BioLegend), anti-human CD3 (APC, APC-65151, Proteintech), anti-human GZMA (PE/Cyanine, 7507221, BioLegend), anti-human CD8a (PE, PE-65144, Proteintech), anti-human GZMB (Pacific Blue, 515407, BioLegend), anti-human CD161 (FITC, FITC-65115, Proteintech), anti-human CD127 (BV510, 563086, BD Bioscience), anti-human CD56 (PE, PE-65067, Proteintech), and anti-human CD16 (PerCP-Cy 5.5, 565421, BD Biosciences).

Techniques: Expressing, Marker, Flow Cytometry, Derivative Assay, Gene Expression, Cell Differentiation

Journal: Journal of Extracellular Vesicles

Article Title: Dynamic Change of PD‐L2 on Circulating Plasma Extracellular Vesicles as a Predictor of Treatment Response in Melanoma Patients Receiving Anti‐PD‐1 Therapy

doi: 10.1002/jev2.70054

Figure Lengend Snippet: PD‐L2 on mucosal melanoma‐derived EVs inhibits T‐cell functions. EVs were isolated from the plasma of melanoma patients in the discovery cohort. Comparison of EV concentrations between clinical benefit (CB) and non‐clinical benefit (NCB) patients at baseline (a) and 4 weeks post‐treatment (b). The proportions of granzyme B+ cells among CD8 T cells in CB versus NCB patients at baseline (c) and 4 weeks post‐treatment (d). The proportions of granzyme B+ cells among CD8 T cells in PD‐L2+ EVs versus PD‐L2‐ EVs at baseline (e) and 4 weeks post‐treatment (f). The proportions of granzyme B+ cells among CD8 T cells in PD‐L1+ EVs versus PD‐L1‐ EVs at baseline (g) and 4 weeks post‐treatment (h). Comparison of proportions of granzyme B+ cells in CD8 T cells after anti‐PD‐1 treatment in the PD‐L2+ EVs treatment group at baseline (i) and 4 weeks post‐treatment (j). (k) Representative flow dot plots of human peripheral CD8 T cells examined for the expression of granzyme B (left), and the proportions of granzyme B+ CD8 T cells are shown on the right ( n = 3 independent biological experiment). (l) Activated PBMC from healthy donors were incubated with EVs or cell medium (CM) containing PD‐L2 or PD‐L1, and IFN‐γ and IL‐2 levels were measured by ELISA ( n = 3 independent biological experiment). (m) Activated Jurkat cells were incubated with EVs or CM containing PD‐L2 or PD‐L1, and IFN‐γ and IL‐2 levels were measured by ELISA ( n = 3 independent biological experiment). All data are presented as the means ± standard deviations. * p < 0.05, ** p < 0.005 and **** p < 0.0005 by a two‐tailed student's t ‐test.

Article Snippet: Pre‐treated samples were stained with a viability dye (TONBO, USA) to exclude dead cells, resuspended in PBS with 2% FBS, and then subjected to antibody incubation at 37°C for 30 min. PBMC and HMVII cells were stained with primary antibodies, including CD3e PerCP‐Cyanine5.5 (650037, Tonbo, USA), CD8 PE (500087, Tonbo, USA) and granzyme B APC/Fire 750 (372209, Biolegend, USA).

Techniques: Derivative Assay, Isolation, Clinical Proteomics, Comparison, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Dynamic Change of PD‐L2 on Circulating Plasma Extracellular Vesicles as a Predictor of Treatment Response in Melanoma Patients Receiving Anti‐PD‐1 Therapy

doi: 10.1002/jev2.70054

Figure Lengend Snippet: PD‐L2‐containing EVs promote tumour growth in mice. (a) The schematic illustrates the process of establishing a syngeneic mouse model by injecting shPDL2 B16‐F10 cells into the flank region of mice. EVs (200 µg) were injected into the tumour ( n = 8 mice per group). Tumour volumes were measured on the indicated day. Growth curve (b) and survival curve (c) with indicated treatments were shown. (d) The proportions of granzyme B+ and Ki‐67+ PD‐1+ cells in CD8 T cells were detected by flow cytometry (FCM; n = 8 independent biological experiment). (e) Representative images of the multiplex immunofluorescence (mIF) staining of DAPI, Ki‐67, CD8 and granzyme B in tumours harvested from the tumour‐bearing mice were displayed, and the positive density of granzyme B+ CD8+ T cells and Ki‐67+ PD‐1+ CD8+ T cells was quantified ( n = 3 independent biological experiment). All data are presented as the means ± standard deviations. * p < 0.05, ** p < 0.005 and **** p < 0.0005 by a two‐tailed student's t ‐test.

Article Snippet: Pre‐treated samples were stained with a viability dye (TONBO, USA) to exclude dead cells, resuspended in PBS with 2% FBS, and then subjected to antibody incubation at 37°C for 30 min. PBMC and HMVII cells were stained with primary antibodies, including CD3e PerCP‐Cyanine5.5 (650037, Tonbo, USA), CD8 PE (500087, Tonbo, USA) and granzyme B APC/Fire 750 (372209, Biolegend, USA).

Techniques: Injection, Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Dynamic Change of PD‐L2 on Circulating Plasma Extracellular Vesicles as a Predictor of Treatment Response in Melanoma Patients Receiving Anti‐PD‐1 Therapy

doi: 10.1002/jev2.70054

Figure Lengend Snippet: PD‐L2‐containing EVs influence tumour response to anti‐PD‐1 treatment in mice model. (a) The study was designed to establish a syngeneic mouse model by injecting shPDL2 B16‐F10 cells into the flank region of mice. EVs (200 µg) were injected into the tumour and anti‐PD‐1 or IgG isotype was injected into the tail vein of mice ( n = 8 mice per group). Growth curve (b) and survival curve (c) of PDL2 EVs or shPDL2 EVs containing tumours treated with anti‐PD‐1 or IgG isotype. (d) The proportion of granzyme B+ CD8 T cells in tumours was detected by flow cytometry (FCM; n = 8 mice per group). (e) Representative images of the multiplex immunofluorescence (mIF) staining of DAPI, CD8 and granzyme B in shPDL2 B16‐F10 tumours with indicated treatment were displayed, and the positive density of granzyme B+ CD8+ T cells was quantified ( n = 3 independent biological experiment). All data are presented as the means ± standard deviations. * p < 0.05, ** p < 0.005 and **** p < 0.0005 by a two‐tailed student's t ‐test.

Article Snippet: Pre‐treated samples were stained with a viability dye (TONBO, USA) to exclude dead cells, resuspended in PBS with 2% FBS, and then subjected to antibody incubation at 37°C for 30 min. PBMC and HMVII cells were stained with primary antibodies, including CD3e PerCP‐Cyanine5.5 (650037, Tonbo, USA), CD8 PE (500087, Tonbo, USA) and granzyme B APC/Fire 750 (372209, Biolegend, USA).

Techniques: Injection, Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy

doi: 10.3389/fimmu.2024.1202017

Figure Lengend Snippet: Antibody panels used for cytotoxic-associated CD4+ T cell phenotypic evaluation.

Article Snippet: At day 10, T cell bulk composition was evaluated via membrane staining with CD3 BV421, CD4 FITC (Diaclone, 954.031.010) and CD8 PE (Diaclone, 854.962.010) antibodies.

Techniques:

Journal: Frontiers in Immunology

Article Title: Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy

doi: 10.3389/fimmu.2024.1202017

Figure Lengend Snippet: Final T cell bulk characterization. T cells are activated, cultured in the presence of IL-2, transduced with HPV16-E7/HLA-DRB1*04-specific TCR-T and selected on the basis of ΔCD19 expression. (A) Graphic representation of experiment design. Figure adapted from images created with BioRender.com . (B) CD4+ and CD8+ T cell transduction efficiency is evaluated through the expression of ΔCD19 selection gene among CD3+ CD4+ or CD3+ CD8+ T cells by flow cytometry; data represent mean+/-SD from 3 independent experiments; two-tailed paired t-test. (C) ΔCD19-based sorting efficiency; data represent mean+/-SD from 4 independent experiments. (D) CD4+ & CD8+ T cell percentage among T cell bulk is evaluated by the expression of CD4 and CD8 co-receptors among CD3+ T cells by flow cytometry; data represent mean+/-SD from 7 independent experiments.

Article Snippet: At day 10, T cell bulk composition was evaluated via membrane staining with CD3 BV421, CD4 FITC (Diaclone, 954.031.010) and CD8 PE (Diaclone, 854.962.010) antibodies.

Techniques: Cell Culture, Transduction, Expressing, Selection, Flow Cytometry, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy

doi: 10.3389/fimmu.2024.1202017

Figure Lengend Snippet: Phenotypic characterization of transduced CD8+ and CD4+ T cells. T cells are activated, cultured in the presence of IL-2, and transduced with HPV16-E7/HLA-DRB1*04-specific TCR-T. (A) Selected transduced (GMTC) and untransduced (UTC) T cells culture-related fold expansion; data represent mean+/-SD from 3 independent experiments; two-tailed paired t-test, *: p< 0.05. (B) Selected transduced and untransduced T cell CD4/CD8 ratio. Data represent individual values and mean from 7 independent experiments; two-tailed paired t-test. (C) Transduced and untransduced, CD4+ and CD8+, T cell activation pattern expression; data represent mean from 3 independent experiments; two-tailed paired t-tests for each subset. (D) Activation score of CD4+ and CD8+ TCR-T cells; data represents individual values and median from 3 independent experiments; two-tailed paired t-test. (E) Transduced and untransduced, CD4+ and CD8+, T cell exhaustion pattern expression; data represent mean+/-SD from 3 independent experiments; two-tailed paired t-test. (F) Exhaustion score of CD4+ and CD8+ TCR-T cells; data represents individual values and median from 3 independent experiments; two-tailed paired t-test.

Article Snippet: At day 10, T cell bulk composition was evaluated via membrane staining with CD3 BV421, CD4 FITC (Diaclone, 954.031.010) and CD8 PE (Diaclone, 854.962.010) antibodies.

Techniques: Cell Culture, Transduction, Two Tailed Test, Activation Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy

doi: 10.3389/fimmu.2024.1202017

Figure Lengend Snippet: In vitro cytotoxic features of transduced CD8+ & CD4+ T cells. T cells are activated, cultured in the presence of IL-2, transduced (and selected) or not with HPV16-E7/HLA-DRB1*04-specific TCR-T, and sorted on the CD4 or CD8 co-receptors expression before being restimulated with HPV16-E7 70-89 -pulsed or not HLA-DRB1*04 BLCL. (A) CD4+ and CD8+ T cell sorting efficiency is evaluated through flow cytometry. Data are representative of 4 independent experiments. (B) Antigen-activated CD4+ and CD8+ T cell CD107a expression is evaluated through flow cytometry. Left: flow cytometry histograms representing CD107a expression of CD8+ or CD4+ T cells after restimulation; light and dark grey histograms corresponding to UTC and GMTC respectively; values obtained from one experiment, representative of 3. Right: bar graph representing CD107a expression of transduced or not CD8+ or CD4+ T cells after restimulation; mean+/-SD from 3 independent experiments; one-tailed paired t-test, *: p< 0.05 and **: p< 0.01. (C) CD4+ and CD8+ T cell-mediated target cell cytolysis is assessed through Annexin V/7AAD co-staining of target cells by flow cytometry. Left: flow cytometry plots representing Annexin V/7AAD expression of peptide-pulsed or not BLCL after co-culture with UTC or GMTC CD4+ or CD8+ T cells; values obtained from one experiment, representative of 3. Right: bar graph representing specific target lysis of transduced or not CD8+ or CD4+ T cells after restimulation; specific target lysis = [(% Annexin V+/7AAD+ cocultured target - % Annexin V+/7AAD+ alone target )/(100 - % Annexin V+/7AAD+ alone target )] x 100; mean+/-SD from 3 independent experiments; one-tailed paired t-test, *: p< 0.05.

Article Snippet: At day 10, T cell bulk composition was evaluated via membrane staining with CD3 BV421, CD4 FITC (Diaclone, 954.031.010) and CD8 PE (Diaclone, 854.962.010) antibodies.

Techniques: In Vitro, Cell Culture, Expressing, FACS, Flow Cytometry, One-tailed Test, Staining, Co-Culture Assay, Lysis

Journal: Frontiers in Immunology

Article Title: Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy

doi: 10.3389/fimmu.2024.1202017

Figure Lengend Snippet: Cytotoxic phenotypic features of ex vivo cultured CD4+ T cells. (A) T cells are activated and cultured in the presence of IL-2 before PMA/iono restimulation and cytotoxicity-associated expression pattern evaluation (panel 1). Figure adapted from images created with BioRender.com . (B) Flow cytometry plots representing CD4+ T cell expression of CD107a, Granzyme B, Perforin, SLAMF7, OX40, 4-1BB, FasL, and TRAIL; data from one experiment, representative of 3. (C) Bar graph representing mean+/-SD from 3 independent experiments for all cytotoxicity-associated CD4+ T cell evaluated markers. (D) T cells are resting or activated and cultured in the presence of IL-2 or IL-7 and IL-15 before PMA/iono restimulation and cytotoxicity-associated expression pattern evaluation (panel 2). Figure adapted from images created with BioRender.com . (E) CD4+ T cell cytotoxicity-associated expression pattern: flow cytometry plots representing CD4+ T cell expression of CD107a, Granzyme B, SLAMF7 after restimulation or not; data from one experiment, representative of 3. (F) Bar graph representing mean+/-SD from 3 independent experiments for all cytotoxicity-associated CD4+ T cell evaluated markers, after restimulation; two-tailed paired t-test, *: p< 0.05 and **: p< 0.01. (G) CD4+ T cells degranulation marker expression intensity, relative to what is observed for their cytotoxic CD8+ T cell counterparts: T cells are either activated and cultured in the presence of IL-2 or IL-7/IL-15 before PMA/iono restimulation and degranulation marker expression intensity (Mean of Fluorescence Intensity or MFI) evaluation, through flow cytometry (panel 2). Bar graph represents mean+/-SD from 3 independent experiments. Pictured statistical analysis are made by comparing the mean of MFI observed for CD4+ and CD8+ T cells, for each culture condition; two-tailed paired t-test, *: p< 0.05 and **: p< 0.01.

Article Snippet: At day 10, T cell bulk composition was evaluated via membrane staining with CD3 BV421, CD4 FITC (Diaclone, 954.031.010) and CD8 PE (Diaclone, 854.962.010) antibodies.

Techniques: Ex Vivo, Cell Culture, Expressing, Flow Cytometry, Two Tailed Test, Marker, Fluorescence

Journal: Frontiers in Immunology

Article Title: Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy

doi: 10.3389/fimmu.2024.1202017

Figure Lengend Snippet: in vivo cytotoxic properties of CD4+ and CD8+ TCR-T cells evaluation (A) Experiment design. Figure adapted from images created with BioRender.com . and Servier Medical ART. (B) Tumor volume measurement for each mouse (green: untreated group, dark blue: total GMTC group, medium blue: CD8+ GMTC, light blue: CD4+ GMTC, dark red: total UTC, medium red: CD8+ UTC, light red: CD4+ UTC), at day 7, 10, 12, 14 and 17. (C) Tumor fold expansion for each mice group (mean+/-SD) (green: untreated group, dark blue: total GMTC group, medium blue: CD8+ GMTC, light blue: CD4+ GMTC, dark red: total UTC, medium red: CD8+ UTC, light red: CD4+ UTC), at day 7, 10, 12, 14, 17; two-tailed unpaired t-test, *: p< 0.05, **: p< 0.01 and ***: p< 0.001. (D) Number of human TIL/10 6 tumor cell count for each mice group (mean+/-SD) (green: untreated group, dark blue: total GMTC group, medium blue: CD8+ GMTC, light blue: CD4+ GMTC, dark red: total UTC, medium red: CD8+ UTC, light red: CD4+ UTC); one-tailed unpaired t-test, *: p< 0.05. (E) TIL phenotypic characterization regarding CD4 (dark pink) or CD8 (blue) co-receptor and transgenic TCR expression or not (full and hatched part of pie charts).

Article Snippet: At day 10, T cell bulk composition was evaluated via membrane staining with CD3 BV421, CD4 FITC (Diaclone, 954.031.010) and CD8 PE (Diaclone, 854.962.010) antibodies.

Techniques: In Vivo, Two Tailed Test, Cell Counting, One-tailed Test, Transgenic Assay, Expressing