Fig 2: Sepsis-derived circulating EVs inhibit T cell function. a: Streaming gate logic diagram. Circle T cell subpopulations in the white blood cell population based on their FSC-A/SSC-A, use of FSC-H/FSC-A to exclude adherent cells; set up of a gate on CD3/CD4 lymphocytes to circle CD3+CD4+ positive cells Group; distinction of negative and positive based on single staining results; b-c: circulating EVs from septic patients interfered with the expression of 24 h surface activation indicators of CD4+ T cells; the CD69 indicator was activated in the early stage and the CD25 indicator in the late stage; d: Proliferation of CD4+ T cells stimulated by circulating EVs at different concentrations (two-way ANOVA); e-f: PD-1 expression after 24 h stimulation of CD4+ T cells by circulating EVs (2.5 µg/ml) in septic patients; g-h: Treg/CD4+ T cell ratio after 24 h stimulation of CD4+ T cells by circulating EVs (2.5 µg/ml) in patients with sepsis; i-l: Expression of cytokines IL-2, IFN-γ, IL-4, and TGF-β in CD4+ T cells after stimulation with circulating EVs (2.5 µg/ml). NC: Normal control group, H-EV: Circulating EVs from healthy humans, S-EV: Circulating EVs from septic patients, sPD-L1: Soluble PD-L1 protein, n = 3, data are presented as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 3: THP-1-derived EV-associated PD-L1 inhibits CD4+ T cell function in LPS. a-b: EVs from cell supernatant interfered with the expression of 24 h surface activation indicators of CD4+ T cells; the CD69 indicator was activated in the early stage and the CD25 indicator in the late stage; c-d: PD-1 expression on CD4+ T after 24 h stimulation with EVs (2.5 µg/ml) from LPS-treated THP-1 cell supernatant; e-f: Treg/CD4+ T cell ratio after 24 h stimulation with EVs (2.5 µg/ml) from LPS-treated THP-1 cell supernatant; g-j: Expression of cytokines IL-2, IFN-γ, IL-4 and TGF-β in CD4+ T cells after stimulation with EVs (2.5 µg/ml) from LPS-treated monocyte supernatant. sPD-L1: Soluble PD-L1 protein, n = 3, data are presented as the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 4: GW4869 intervention in sepsis restored T cell function by targeting monocytes. a: Cartoon depicting the transwell setup. Monocytes were seeded in the upper chamber and CD4+ T cells cultured in the lower chamber. LPS or GW4869 was added to the upper chamber. Transwell insert enabled EVs to pass through the membrane into the lower chamber; b: After GW4869 treatment, 100 ml of cell supernatant was collected to extract EVs that were then resuspended in an equal volume of PBS. EVs protein levels were subsequently measured using the BCA assay (mean ± SEM); c: Western blot was used to detect the expression of EV markers after GW4869 treatment; d-e: Expression of CD69 and CD25 on CD4+ T cells after LPS and GW4869 treatment; f-g: The expression of PD-1 on CD4+ T cells after 24 h of co-culture with LPS and GW4869 treatment (mean ± SEM); h-i: Treg/CD4+ T cell ratio after 24 h stimulation of co-culture with LPS and GW4869 treatment (median ± IQR); j-m: Expression of IL-2, IFN-γ, IL-4, and TGF-β in CD4+ T cells after 24 h of co-culture with LPS and GW4869 treatment (mean ± SEM). n = 3, *P < 0.05, **P < 0.01