Journal: bioRxiv

Article Title: Combination antagonism of TNF superfamily signaling for T cell immunosuppression

doi: 10.64898/2026.04.27.721101

Figure Lengend Snippet: ( A ) Interleukin-2 (IL-2) and interferon gamma (IFN-γ) inhibition in CD3 + cells during allogeneic one-way MLR measured at day 2 and day 5, respectively, treated with 50 nM combination pharmacological inhibitors of TNF/TNFR targets of interest. Monotreatment groups included 50 nM of human IgG1. Cytokine inhibition was calculated as percent reduction from IgG1 control treatment. Ill denotes P <0.05 in student t-test for monotreatments vs. IgG1 control, # denotes P <0.05 in student t-tests for combination treatment groups vs. IgG1 controls and both constituent monotreatments. ( B ) Normalized IL-2 and IFN-γ expression in four promising combinations of TNF/TNFR inhibitors. ( C-D ) Proliferation in CD3 + cells during allogeneic one-way MLR measured at day 5 in the presence of combination pharmacological inhibition of TNF/TNFR targets of interest.* denotes P <0.05 in student t-test. Data from three unique stimulator-responder MLR pairs, mean ± SEM plotted. Large-molecule pharmacological TNF/TNFR inhibitors used: Adalimumab (abbr. Ada, α-TNF), Pateclizumab (α-LTA), Baminercept (abbr. Bam, LTβR decoy), Dapirolizumab (abbr. Dapiro, α-CD40L), Amlitelimab (abbr. Amlite, α-OX40L), CD30Li (α-CD30L), 4-1BB-Fc (4-1BB decoy), Quisovalimab (α-LIGHT), Duvakitug (α-TL1A), and GITRi (α-GITR).

Article Snippet: Adalimumab (Cat. HY-P9908), Pateclizumab (Cat. HY-P990034), Baminercept (Cat. HY-P99459), Amlitelimab (Cat. HY-P99434), Dapirolizumab (Cat. HY-P99842A), Quisovalimab (Cat. HY-P99810), Duvakitug (Cat. HY-P99842A), human IgG1 (Cat. HY-P99001) and human IgG4 (Cat. HY-P99003) were purchased from MedChemExpress.

Techniques: Inhibition, Control, Expressing

Journal: bioRxiv

Article Title: Combination antagonism of TNF superfamily signaling for T cell immunosuppression

doi: 10.64898/2026.04.27.721101

Figure Lengend Snippet: ( A-B ) Interleukin-2 (IL-2) and interferon gamma (IFN-γ) inhibition in CD3 + cells during allogeneic one-way MLR measured at day 2 and day 5, respectively, treated with 50 nM combination pharmacological inhibitors of TNF/TNFR targets of interest. Monotreatment groups included 50 nM of human IgG1. Cytokine expression normalized to IgG1 control treatment. * denotes P <0.05 in student t-test for monotreatments vs. IgG1 control. Data from three individual stimulator-responder MLR pairs, mean ± SEM plotted.

Article Snippet: Adalimumab (Cat. HY-P9908), Pateclizumab (Cat. HY-P990034), Baminercept (Cat. HY-P99459), Amlitelimab (Cat. HY-P99434), Dapirolizumab (Cat. HY-P99842A), Quisovalimab (Cat. HY-P99810), Duvakitug (Cat. HY-P99842A), human IgG1 (Cat. HY-P99001) and human IgG4 (Cat. HY-P99003) were purchased from MedChemExpress.

Techniques: Inhibition, Expressing, Control

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A ) Expression of CD27 and IgD on CD19 + B cells. Right: Percentage of IgD + CD27 − naïve B cells and IgD − CD27 − DN B cells. ( B ) Expression of CD27 and CD38 on CD19 + B cells. Right: Percentages of CD27 hi CD38 hi 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) to (D)] HC ( n = 63), irAE ( n = 33), RAC ( n = 46), and ICI ( n = 20). ( E to 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 plots of the citrullinated or noncitrullinated relative IgG or IgM isotype autoantigen levels comparing RA versus HC, irAE versus ICI, irAE versus HC, or irAE versus RA. The autoantigens were labeled when P < 0.01. TNF-α reactivities resulted from the administration of anti–TNF-α therapy for the treatment of RA. ( I ) Immunoglobulin isotype levels in the plasma were measured by multiplex assay. HC ( n = 22), irAE ( n = 34), RAC ( n = 46), and ICI ( n = 26). ( J ) CXCL13 levels in the plasma were measured by enzyme-linked immunosorbent assay (ELISA). HC ( n = 20), irAE ( n = 34) RAC ( n = 47), and ICI ( n = 23). ( K ) B-cell activating factor (BAFF) levels in the plasma were measured by multiplex assay. HC ( n = 21), irAE ( n = 34), RAC ( n = 47), and ICI ( n = 18). Data in graphs represent mean ± SEM. Significance was tested by one-way ANOVA [(A) to (D) and (I) to (K)] and logistic regression (H). [(A) to (D) and (F) to (K)] ICI, ICI control.

Article Snippet: Pembrolizumab (100 μg/ml; Keytruda) or isotype control IgG4 (Bio X Cell, catalog no. 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, Control

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A to C ) Human naïve B cells were isolated from PBMCs and cultured under indicated conditions for 7 days; pembrolizumab or isotype control IgG4 (IgG4 Iso) was added on day 2; n = 8. (A) Expression of CD38 and CD27 on B cells. Right: Percentages of CD27 + CD38 − and 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) and (B) by multiplex assay. ( D to F ) B cells from Humanized PD-1 (HuPD-1) mice were isolated and cultured with LPS, IL-4, BAFF, or ODN 2006, anti-IgM, IL-21, IL-4 or R848, anti-IgM, anti-CD40, IL-21, and IFN-γ for 3 days; pembrolizumab or isotype control was added on day 1. (D) Expression of IgG2c on activated B cells. FSC-H, Forward scatter height. 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 were measured by multiplex assay; n = 5. Data in graphs represent mean ± SEM. Significance was tested by two-way ANOVA.

Article Snippet: Pembrolizumab (100 μg/ml; Keytruda) or isotype control IgG4 (Bio X Cell, catalog no. 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, Multiplex Assay

Journal: Science Advances

Article Title: Inflammatory arthritis irAE may represent a unique autoimmune disease primarily driven by T cells but likely not autoantibodies

doi: 10.1126/sciadv.aea4262

Figure Lengend Snippet: ( A to G ) PBMCs from the patients with irAE were cultured in the plate coated with anti-CD3 and anti-CD28 (10 μg/ml) in the presence of IgG1 isotype control or anti–human IL-6R (50 μg/ml), anti–human IL-12p40 (50 μg/ml), and anti–human IFNAR1 (50 μg/ml) for 3 days; n = 9. (A) Expression of CD38 and CD127 on CD8 + T cells. Right: Percentage of CD38 + CD127 − CD8 + T cells. (B and C) CD8 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (B) Expression of granzyme B and IFN-γ. Right: Percentage of granzyme B + IFN-γ + CD8 + T cells. (C) Expression of perforin and IFN-γ. Right: Percentage of perforin + IFN-γ + CD8 + T cells. [(D) and (E)] MFIs of GluCy5 (D), TMRM, and MTDR (E) in CD8 + T cells were presented. [(F) and (G)] CD4 + T cells activated for 5 days were restimulated with PMA, ionomycin, and monensin for 5 hours. (F) Expression of IL-21 and IL-2. Right: Percentage of IL-21 + IL-2 + CD4 + T cells. (G) 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 paired Student’s t test [(A) to (G)].

Article Snippet: Pembrolizumab (100 μg/ml; Keytruda) or isotype control IgG4 (Bio X Cell, catalog no. CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Cell Culture, Control, Expressing

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