Fig 1: USCs induce T cell apoptosis in beads activated condition. The effect of USCs on cytokine production and T cell viability was measured after 48 hours and 5 days of coculture respectively. A, PI staining of CD3+ T cells population from PBMCs cultured alone vs cocultured with USCs (99.1 ± 0.7% vs 98.3 ± 0.6%), or in the presence of anti CD3/CD28 beads (93.5 ± 0.3% vs 82.5 ± 4.9%). Viability of beads‐stimulated T cells significantly decreased in the presence of USCs. B, Annexin V staining for apoptotic CD3+ T cells of PBMC cultured alone vs in USCs coculture (1.5 ± 0.17 vs 5.1 ± 1.2%), or in the presence of anti CD3/CD28 beads (8.5 ± 1.3 vs 13.0 ± 1.5%), showing the increase of apoptotic cells cultured under activated condition, which significantly increases in the presence of USCs. C, Representative sample of Annexin/Live Dead double staining of CD3+ T cells population from PBMCs cultured either alone or with untreated USCs, or with anti CD3/CD28 beads. Double negative are live cells, Annexing V positive Life/Dead negative are apoptotic cells and double positive necrotic cells. D, The effect of USCs on PBMCs cytokine production in response to anti‐CD3/CD28 beads measured by beads array shows that USCs significantly suppressed the production of the inflammatory cytokines, TNF‐α, TNF‐β and IL‐13 by T cells in response to anti‐CD3/CD28 stimulation, while significantly increasing Il‐6, BAFF and APRIL. Values are presented as mean ± SEM (A, B, D; n = 3). P values were determined by RM One‐way ANOVA, with Geisser–Greenhouse correction followed by Tukey multiple comparison. *P < .05, **P < .01, ***P < .001, ****P < .0001
Fig 2: USCs secrete cytokines known to modulate B cells. USCs and BM‐MSCs were cultured physically separated from PBMC in Trans‐well and the B population was analyzed for its proliferation. To elucidate on B cell stimulatory secreted factors, PBMC, USCs coculture, BM‐MSCs coculture and USCs supernatants were analyzed for B cell related cytokines. A, Proliferation analysis of the B cells from PBMC cultured in Trans‐wells (T) either alone, or over either BM‐MSCs (T) (3.45 ± 1.3 vs 4.7 ± 1.2%) or USCs (T), or in direct contact with USCs (24.6 ± 6.7 vs 57.2 ± 6.8%) (n = 5). B, Analysis of cytokines concentration in supernatants collected from PBMCs cultured either alone or with BM‐MSCs or USCs for possible B cell stimulants showing significant higher BAFF and APRIL concentration in USCs coculture. C, Heat map presenting log 10 B cell related cytokines concentration (Z‐score normalized by row) in supernatants collected from USCs, PBMCs and USCs + PBMC. D, Analysis of the B cell related cytokines concentration in supernatants collected from PBMC compared to USCs cocultured showing significant increase in TNFα, IL10, IL6, BAFF, APRIL, and CD40L in the presence of USCs. E, Cytokines concentration in supernatant collected from 105 USCs, cultured for 48 hours. F, Cytokines concentration in supernatant collected from 105 BM‐MSCs, cultured for 48 hours (n = 3). Values are presented as mean ± SEM. B,D, Cytokines are presented after log treatment of the data. P values were determined by One‐way ANOVA (A,B) or paired t test (D), *P < .05, **P < .01, ***P < .001, ****P < .0001
Fig 3: Levels of monocytes correlated positively with the TNF-α and IL-6 plasma concentrations in PDAC patients. Levels of monocytes correlated to the concentration of TNF-α (A), IL-6 (B), and IL-10 (C) in the plasma of PDAC patients.
Fig 4: Expression of CD71 on neutrophils correlated positively with the IL-6, IFN-γ, CD40-L and BAFF plasma levels in PDAC patients. Levels of IL-6 (A), IFN-γ (B), TNF-α (C), IL-10 (D), CD40-L (E) and B-cell activating factor (BAFF) (F) in the plasma of clinical control (n = 5) and PDAC patients (n = 17) and the correlation to the MFI of CD71 on circulating neutrophils; *p < 0.05.
Fig 5: Functional role of monocytes during pancreatic cancer. (A) Schematic diagram denoting coculture system (Created in BioRender. Kuchenreuther, I. (2025) https://BioRender.com/e51y219 accessed on 19 February 2025). (B) Expression of HVEM on different monocyte subsets. (C) Expression of CD206 on different monocyte subsets. (D) Expression of CD163 on different monocyte subsets. (E) Cytokine levels of IL-6, TNF-a and IL-10 in monocytes with and without Panc-1. * p < 0.05; ** p < 0.006; PBMC—peripheral blood mononuclear cells; CM—classical monocytes; IMM—intermediate monocytes; NCM—non-classical monocytes; IL-6—interleukin 6; TNFa—tumor necrosis factor alpha; IL10—interleukin 10.
Supplier Page from BioLegend for LEGENDplex™ Human B Cell Panel (13-plex) with V-bottom Plate