Journal: NPJ Breast Cancer

Article Title: TGF-α/EGFR-mediated lymphatic metastasis reveals a repositionable therapeutic target in breast cancer

doi: 10.1038/s41523-026-00941-0

Figure Lengend Snippet: Studies were performed to analyze whether blockade of TGF-α could inhibit chemotactic invasion and lymphatic metastasis of 4T1-EGFR cells. a Results from transwell invasion assays showing that Fepixnebart, a neutralizing antibody against TGFα (aTGFα), significantly inhibited chemotactic invasion of 4T1-EGFR cells toward svLEC-conditioned media. An isotype matched IgG antibody was used as control. For each condition, three fields per well were imaged and quantified across three independent experiments based on crystal violet-stained areas. Statistical analysis was performed using an unpaired two-tailed Student’s T-test. b Schematic overview of the in vivo experimental setup. 4T1 cells were orthotopically implanted into mice, followed by tail vein injection of Fepixnebart. Tumor-draining lymph nodes were collected for analysis 10 days later. c Bar graphs showing quantification of CK⁺ tumor cells in inguinal (ILN) and axillary (ALN) lymph nodes 10 days after orthotopic injection of 4T1-EGFR cells into mammary fat pads. Mice received a single intravenous injection of Fepixnebart (10 mg/kg; n = 9) or isotype IgG control ( n = 8) at the time of tumor cell implantation. Data are shown as mean ± SEM; statistical analysis by unpaired two-tailed Student’s t-test.

Article Snippet: Simultaneously, mice received a single intravenous dose of Fepixnebart (10 mg/kg) or an isotype-matched human IgG4 control antibody (HY-P99003, MedChemExpress).

Techniques: Control, Staining, Two Tailed Test, In Vivo, Injection

Journal: Frontiers in immunology

Article Title: Analysis of the mechanisms regulating soluble PD-1 production and function in human NK cells.

doi: 10.3389/fimmu.2023.1229341

Figure Lengend Snippet: FIGURE 1 Characterization of the isolated recombinant sPD-1 protein. (A) Gel-based proteomic analysis of purified sPD-1. Left panel: Coomassie staining of the isolated recombinant sPD-1 revealed a band of approximately 20 kDa corresponding to sPD-1 molecular weight. BSA sample was used as a control. Right panel: western blot analysis allowed detection of the recombinant sPD-1 protein with both the anti-Streptavidin-HRP and anti-PD-1 antibodies. (B) SKNAS and IMR-32 cell lines were analysed for PD-L1 and PD-L2 expression. Only PD-L1 could be detected on the surface of both cell lines. (C) Analysis of recombinant sPD-1 binding to PD-L1 ligand. Flow cytometry analysis demonstrated that recombinant sPD-1 was able to bind PD-L1 expressed by SKNAS and IMR-32 cell lines. (D) SKNAS were incubated with PBS, IgG1 Isotype or Atezolizumab and then analysed for PD- L1 expression. Abrogation of PD-L1 signal occurred only when tumor cells were treated with Atezoliumab (left panel). Binding of sPD-1 to tumor expressed PD-L1 was abrogated when NB cells were previously incubated with Atezolizumab. (E) Incubation of PD-L1+ SKNAS and IMR-32 cell lines with recombinant sPD-1 resulted in a decreased PD-L1 signal. MFI from ctrl (not stained) samples was subtracted from their corresponding stained samples. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **; p<0.05 *.

Article Snippet: Binding of Nivolumab (A2002, Selleckchem) to PD-1 was performed incubating stimulated NK cells with IgG4 control Isotype (DDXCH04P-100, Novus Biological) or Nivolumab for 30 min at 4°C and then with anti-PD1 for 30 min at 4°C.

Techniques: Isolation, Recombinant, Staining, Molecular Weight, Control, Western Blot, Expressing, Binding Assay, Flow Cytometry, Incubation

Journal: Frontiers in immunology

Article Title: Analysis of the mechanisms regulating soluble PD-1 production and function in human NK cells.

doi: 10.3389/fimmu.2023.1229341

Figure Lengend Snippet: FIGURE 2 Recombinant sPD-1 can modulate NK cell effector function. (A) Control and stimulated NK cells were analysed for membrane PD-1 and PD-L1 expression. Membrane-bound PD-1 was detected only in stimulated NK cells compared to control cells. Conversely, while Ctrl NK cells expressed PD-L1, it was barely detectable on stimulated NK cells. One representative experiment has been shown. Quantifications of four different experiments have been reported. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **; p< 0.001 ***. (B) NB cell lines were incubated with stimulated NK cells, expressing PD-1, with or without recombinant sPD-1. An increase in cytotoxicity toward both SKNAS (n=7) and IMR-32 (n=8) cell lines at different Effector (E) Target (T) ratios was observed only when recombinant sPD-1 was present in the co-culture. Data have been compared using paired T test, p< 0.05 *; p< 0.01 **, p< 0.001 ***. (C) Similarly, in the presence of recombinant sPD-1 an increase in degranulation (left and middle panels) and accumulation of IFN-g (right panel) of NK cells were observed. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **, p< 0.001 ***. (D) Comparison of sPD-1 and Nivolumab treatments on cytotoxicity of stimulated NK cells. Incubation with Nivolumab abrogated PD-1 signal compared to Ctrl and IgG4 samples (left panel). Increase of cytotoxicity toward SKNAS cell line at different E:T ratio was observed in both the analysed conditions (treated or not with Nivolumab) only in the presence of sPD-1. Values are mean ± SEM. Data have been compared using paired T test, p< 0.05 *; p< 0.01 **, p< 0.001 ***.

Article Snippet: Binding of Nivolumab (A2002, Selleckchem) to PD-1 was performed incubating stimulated NK cells with IgG4 control Isotype (DDXCH04P-100, Novus Biological) or Nivolumab for 30 min at 4°C and then with anti-PD1 for 30 min at 4°C.

Techniques: Recombinant, Control, Membrane, Expressing, Incubation, Co-Culture Assay, Comparison

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: T cells isolated from healthy human donors were activated with anti-CD3/28 beads and IL-2 was added on Day 3. A PD-1, PD-L1, and PD-L2 expression were measured via flow cytometry over 15 days for N = 8 in CD4+ and N = 5 in CD8 + T cells. B Day 4 purified CD8 + T cells were restimulated with autologous irradiated PBMC and anti-CD3 (1 μg/mL) ± anti-CD28 (1 μg/mL) in the presence of control antibody, anti-PD-1, or anti-PD-L1 at 10 μg/mL for 20 hr. Death was assessed via flow cytometry by propidium iodide staining. Single dot correlates with one human donor. Connecting line compares change in untreated and treated samples within the same donor. N = 3. C Diagram illustrating the proxy APC (pAPC) design created with a 1:1:1 ratio of anti-CD3, anti-CD28, and IgG1κ or recombinant human Fc-chimera PD-L1. D Freshly isolated CD4+ or CD8+ human T cells were activated at a 1:1 cell:bead ratio with IgG or PD-L1 beads and monitored via flow cytometry for CD69 expression after 24 hr ( N = 3 CD4 + , N = 5 CD8 + ) and CD25 expression after 72 hr ( N = 3 CD4 + , N = 6 CD8 + ). E Blastogenesis patterns of unactivated, IgG, or PD-L1 activated cells on Day 3 are demonstrated by dot plots assessing forward scatter (FSC) (x-axis) and side scatter (SSC) (y-axis). The progressive changes in FSC monitored via flow cytometry over 3 days are summarized for CD4+ and CD8 + T cells. N = 3 CD4 + , N = 5 CD8 + .

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Isolation, Expressing, Flow Cytometry, Purification, Irradiation, Control, Staining, Recombinant

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A CD8+ or CD4 + T cells were restimulated on Day 4 or Day 11 with p328-IgG of p328-L1 beads at a 1:1 bead:cell ratio and monitored for cell loss via flow cytometry 24 hr later using propidium iodide. N = 8 CD4+ early, N = 11 CD4+ late, N = 9 CD8+ early, N = 18 CD8+ late. B The difference in % cell loss between p328-IgG and p328-L1 beads in ( A ) is plotted for each donor, cell type, and cell stage evaluated. (same N as ( A ). C RICD assays set-up in ( A ) were examined on the flow cytometer using propidium iodide to calculate the percentage of live cells remaining after 24 hr of restimulation. N = 7 CD4+ early, N = 9 CD4+ late, N = 12 CD8+ early, N = 8 CD8+ late. D PD-L1 dose response was determined using the standard RICD setup amd a new bead set that gradually decreased PD-L1 concentration on the bead from 33% to 0%. Day 4 CD4+ and CD8 + T cells were utilized in this experiment. N = 12 CD4 + , N = 10 CD8 + , statistical analysis was calculated with two-way ANOVA. E Diagram representing RICD setup and creation of secondary bead set that was ligated 100% to IgG1κ (control secondary bead) or 67% IgG1κ and 33% recombinant PD-L1, matching the PD-L1 protein concentration on the p328-L1 primary bead. F Day 4 CD8+ and CD4 + T cells were restimulated with a 2:1 bead:cell ratio comprising a 1:1 ratio of the primary bead and 1:1 ratio of the secondary bead. RICD was monitored as in ( A ) N = 12.

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Flow Cytometry, Concentration Assay, Control, Recombinant, Protein Concentration

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A Representative histogram of data displaying demarcation of AnnexinV hi cells -/+ bead restimulation. B Day 4 and Day 11 CD4+ and CD8 + T cells were left unstimulated or were restimulated with p328-IgG or p328-L1 beads for 6 hr before being stained with AnnexinV and propidium iodide and read on the flow cytometer. N = 6. C Representative flow cytometric histogram of PI cell cycle phases based on DNA content. D Day 4 and Day 11 CD4+ and CD8 + T cells were left unstimulated or were restimulated with p328-IgG or p328-L1 beads for 18 hr before being permeabilized and stained with propidium iodide to determine % of cells in the subG1 phase. N = 8 CD4+ early, N = 8 CD4+ late, N = 10 CD8+ early, N = 9 CD8+ late. E Analysis using gated cells from ( D ) was performed to quantify changes in the G1 phase. F Cell phase characterization from ( D ) was analyzed to determine the PD-L1 induced difference in subG1 and G1 phases.

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A CD8+ or CD4 + T cells were restimulated on Day 4 with soluble anti-CD3 (OKT3) at 100 ng/mL or anti-CD3/CD28 coated beads at a 1:1 bead:cell ratio. Cell loss/apoptosis was quantified 18–24 hr later after propidium iodide staining and flow cytometry. N = 5 CD4+ early, N = 6 CD4+ late, N = 5 CD8+ early, N = 6 CD8+ late. B Day 4 and Day 11 CD4+ and CD8 + T cells from N = 5 individual donors were stained for CD28 and PD-1 expression and compared via flow cytometry. C Diagram illustrating restimulation beads conjugated with or without anti-CD28, with p328-IgG used to maintain total protein concentration. D CD8+ or CD4 + T cells were restimulated on Day 4 with beads illustrated in ( C ) at a 1:1 bead:cell ratio, monitored for cell loss 24 hr later using propidium iodide using flow cytometry. E The difference in cell loss induced by restimulation with beads -/+ anti-CD28 (leaving anti-CD3 + /- PD-L1-Fc constant) was plotted for every donor ( N = 10).

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Staining, Flow Cytometry, Expressing, Protein Concentration

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A Day 4 CD4+ and CD8 + T cells ( N = 4 donors each) were restimulated with p328-IgG or p328-L1 beads at a 1:1 bead:cell ratio for 5 hr prior to lysate collection. Control cells were left unstimulated or treated with 1 μM STX. After cell lysis and protein standardization, donor lysates were pooled for each stimulation condition and analyzed on the apoptosis protein array. Representative spots for differentially expressed proteins are shown. Protein signal intensity from membrane imaging was analyzed and normalized using QuickSpots software. T cells were restimulated as in ( A ), lysed in RIPA buffer and separated by SDS-PAGE to quantify FASL ( B ) and survivin ( C ). Data are representative of experiments for 3–4 separate donors. ( D ) Day 4 CD4+ and CD8 + T cells were treated with 10 μg/mL anti-FASL blocking antibody for 1 hr prior to restimulation with the quad bead set. Cells were restimulated for 24 hr and stained with propidium iodide; % viability was assessed by flow cytometry. N = 7 CD4 + , N = 5 CD8 + .

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Control, Lysis, Protein Array, Membrane, Imaging, Software, SDS Page, Blocking Assay, Staining, Flow Cytometry

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Humoral immunity remains largely intact in irAE patients, while seronegative RA patients have increased atypical B cell frequency and autoantibodies. (A) Expression of IgM and IgD on CD27 - CD24 - CD38 - B cells. Right, percentage of IgD + CD27 - CD24 - CD38 - naïve B cells and IgM - IgD - CD27 - CD24 - CD38 - double-negative B cells. (B) Expression of CD27 and CD38 on CD19 + B cells. Right, percentages of CD27 hi CD38 hi antibody-secreting cells (ASCs) and CD27 + CD38 - memory B cells. (C) Expression of CD138 on CD27 hi CD38 hi ASCs. Right, percentage of CD138 + ASCs and CD138 - ASCs. (D) Expression of CD11c and CD21 on CD19 + B cells. Right, percentage of CD11c + CD21 - CD19 + B cells. A-D, HC (n = 63), irAE (n = 33), RAC (n = 46) ICI, (n = 20). (E-G) GSEA was performed on the B cells between irAE and ICI. (E) Significantly enriched pathways in B cells from irAE and ICI. GSEA plots of IFN-α and IFN-γ response (F), and oxidative phosphorylation (G). (H) The volcano plot of the citrullinated or non-citrullinated relative IgG or IgM-isotype autoantigen levels comparing RA vs HC, irAE vs ICI, irAE vs HC, or irAE vs RA. The autoantigens were labeled when p < 0.01. (I) Immunoglobulin isotype levels in the plasma were measured by multiplex assay, HC (n = 22), irAE (n = 34), RAC (n = 46), ICI (n = 26). (J) CXCL13 levels in the plasma were measured by ELISA, HC (n = 20), irAE (n = 34) RAC (n = 47), ICI (n = 23). (K) BAFF levels in the plasma were measured by multiplex assay, HC (n = 21), irAE (n = 34), RAC (n = 47), ICI (n = 18). Data in graphs represent mean ± SEM, Significance was tested by one-way ANOVA (A-D, I-K), logistic regression (H).

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Expressing, Phospho-proteomics, Immunopeptidomics, Labeling, Clinical Proteomics, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Pembrolizumab does not substantially affect B cell activation and antibody production ex vivo (A-C) Human naïve B cells were isolated from PBMCs and cultured under indicated conditions for 7 days; Pembrolizumab or isotype control IgG4 was added on Day 2, n = 8. (A) Expression of CD38 and CD27 on B cells. Right, percentages of CD27 + CD38 - , CD27 + CD38 + B cells. (B) Expression of CD138 on CD27 + CD38 + B cells. Right, percentage of CD138 + CD27 + CD38 + B cells. (C) Different immunoglobulin isotypes were measured in the culture supernatant of (A-B) by multiplex assay. (D-F) B cells from HuPD-1 mice were isolated and cultured with LPS, IL4, BAFF, or ODN2006, anti-IgM, IL-21, IL-4 or R848, anti-IgM, anti-CD40, IL-21, IFN-γ for 3 days, pembrolizumab or isotype control was added on Day 1. (D) Expression of IgG2c on activated B cells. Right, percentage of IgG2c + B cells, n = 5. (E) Expression of IgG1 on activated B cells. Right, percentage of IgG1 + B cells, n = 5. (F) Different immunoglobulin isotypes in the supernatant of were measured by multiplex assay, n = 5. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Activation Assay, Ex Vivo, Isolation, Cell Culture, Control, Expressing, Multiplex Assay

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: PD-1 inhibition does not have substantial impact on B cells ex vivo (A) Human naïve B cells were isolated from healthy donor PBMCs and stimulated with different conditions; Pembrolizumab (Keytruda) or isotype control IgG4 was added into the culture media on day 2, mean fluorescence intensity (MFI) of CD86 on B cells was measured, n = 8. (B-F) B cells were isolated from HuPD-1 mice, labeled with CTV, and cultured under 3 different conditions: LPS, rmIL-4, BAFF or Anti-IgM, CpG ODN, rmIL-21, rmIL-4, 100 rhIL-2 or R848, anti-CD40, anti-IgM, rmIL-21, rmIFN-γ for 3 days. Pembrolizumab or isotype control IgG4 was added on day 1, n = 5. (B) Summary of human PD-1 MFI on B cells from different groups. (C) Expression of CD138 on activated B cells. Right, percentage of CD138 + B cells. (D) Summaries of activation marker CD69 and CD86 MFIs on B cells from different groups. (E) Summaries of CD71 and CD98 MFIs on B cells from different groups. (F) Representative flow cytometry plot of CTV dilution on B cells. Right, a summary of the proliferation index of B cells from different groups. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Inhibition, Ex Vivo, Isolation, Control, Fluorescence, Labeling, Cell Culture, Expressing, Activation Assay, Marker, Flow Cytometry

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Inflammatory cytokines enriched in irAE patients promote both CD4 + and CD8 + T cell functions (A) Summaries of the percentage of CD45RA - CD8 + T cells at day 1 or day 5 of the culture, n = 6. (B) Summary of the percentage of Ki67 + in CD45RA - CD8 + T cells, n = 4. (C) Expression of CD69 on cultured CD8 + T cells at day 1. Right, CD69 mean fluorescence intensity (MFI) relative to the control in each experiment, n = 5. (D) Summary of the CD25 MFI relative to the control in each experiment, n = 5. (E) Summary of the percentage of Granzyme B + CD8 + T cells, n = 5. (F) Summary of the percentage of CD45RA - CD4 + T cells at day 5 of the culture, n = 4. (G) Summary of the percentage of Ki67 + CD45RA - CD4 + T cells, n = 4. (H-L) CD4 + T cells were isolated from healthy donor PBMCs, stimulated with 10 μg/mL plate-coated anti-human CD3 and anti-human CD28 in the presence of vehicle control (control), 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, or the combination of them for 5 days, followed by restimulating with PMA/Ionomycin/Monensin for 5 h. (H) Expression of TNF-α in CD45RA - CD4 + T cells. Right, percentage of TNF-α + cells in CD45RA - CD4 + T cells, n =4. (I) Expression of IL-2 and IL-4 in CD45RA - CD4 + T cells. Right, percentage of IL-4 + and IL-2 + cells in CD45RA - CD4 + T cells, n = 3. (J-L) Summaries of the percentages of TNF-α + (J), IL-4 + (K), or IL-2 + (L) cells in CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells at day 5, n = 4. (M-Q) PBMCs from irAE patients were stimulated with 10 μg/mL plate-coated anti-human CD3 and anti-human CD28 in presence of IgG1 isotype control or a combination of 50 μg/mL anti-human IL-6R, 50 μg/mL anti-human IL-12p40 and 50 μg/mL anti-human IFNAR1 for 3 days, n = 9. (M) Expression of granzyme B and perforin in CD8 + T cells. Right, percentage of granzyme-B + perforin + CD8 + T cells. (N) MFI of MTG in CD8 + T cells. (O, P) MFI of GluCy5 (O), TMRM and MTDR (P), and MTG (Q) in CD4 + T cells. Data in graphs represent mean ± SEM, Significance was tested by One-way ANOVA (A-L), and paired Student’s T-test (N-Q).

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Expressing, Cell Culture, Fluorescence, Control, Isolation

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Inflammatory cytokines enriched in irAE patients promote both CD4 + and CD8 + T cell functions (A-G) CD8 + T cells were isolated from healthy donor PBMCs and cultured in the plate coated with 10 μg/mL anti-human CD3 and anti-human CD28 for indicated days in the presence of vehicle control (control), 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, or the combination of IFN-α, IL-6 and IL-12. (A) Summaries of the percentage of CD38 + CD127 - CD8 + T cells at day 1 and day 5, n = 5. (B) CD69 expression was measured on day 5, and the mean fluorescence intensity (MFI) of CD69 was summarized on the right, n = 4. (C) CD25 expression was measured on day 5, and the MFI of CD25 was summarized on the right, n = 4. (D) CD8 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, expression of granzyme B and IFN-γ in CD8 + T cells were examined by flow cytometry. Right, percentage of granzyme B + IFN-γ + CD8 + T cells, n = 5. (E-G) CD8 + T cells were cultured for 5 days. MFIs (all relative to those in control condition) of MTDR (E), MTG (F), and GluCy5 (G) were summarized, n = 5. (H-L) CD4 + T cells were isolated from the PBMCs of HC, cultured in the plate coated with 10 μg/mL anti-human CD3 and anti-human CD28 for indicated days in the presence of 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, control or the combination of them. (H) CD4 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, and IL-21 level was examined in the CD45RA - CD4 + T cells, n =3. (I) Activated CD4 + T cells were restimulated with 10 μg/mL plate-coated anti-human CD3 and anti-human CD28 with monensin for 6 h, and expression of CXCL13 was measured in CD45RA - CD4 + T cells, n = 4. (J) CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells were examined at day 5, and the percentages were summarized, n = 4. (K) Percentage of CXCL13 + cells in CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells at day 5, n = 4. (L) Percentage of IL-21 + cells in CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells at day 5, n = 3. (M-S) PBMCs from the irAE patients were cultured in the plate coated with 10 μg/mL anti-CD3 and anti-CD28 in the presence of IgG1 isotype control or 50 μg/mL anti-human IL-6R, 50 μg/mL anti-human IL-12p40 and 50 μg/mL anti-human IFNAR1 for 3 days, n = 9. (M) Expression of CD38 and CD127 on CD8 + T cells. Right, percentage of CD38 + CD127 - CD8 + T cells. (N, O) CD8 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, (N) Expression of Granzyme B and IFN-γ. Right, percentage of granzyme B + IFN-γ + CD8 + T cells, (O) Expression of perforin and IFN-γ. Right, percentage of perforin + IFN-γ + CD8 + T cells. (P, Q) MFIs of GluCy5 (P), TMRM, and MTDR (Q) in CD8 + T cells were presented. (R, S) CD4 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, (R) Expression of IL-21 and IL-2. Right, percentage of IL-21 + IL-2 + CD4 + T cells. (S) Expression of CD38 and CXCR5 on CD4 + T cells. Right, percentage of CD38 + CXCR5 - CD4 + T cells. Data in graphs represent mean ± SEM, Significance was tested by One-way ANOVA (A-L), and paired Student’s t-test (M-S).

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Isolation, Cell Culture, Control, Expressing, Fluorescence, Flow Cytometry