Red blood cell lysate

RAW264.7 cells were divided into three groups (50 μL/sample): blank group, isotype control group, and experimental group. The isotype control group received 1 μL Rat IgG2a Kappa‐FITC, 1 μL Rat IgG2b Kappa‐PerCP‐Cy5.5, and 1 μL Rat IgG2a Kappa‐APC. The experimental group received 1 μL CD86 monoclonal antibody‐FITC, 1 μL CD11b monoclonal antibody‐PerCP‐Cy5.5, and 1 μL F4/80 monoclonal antibody‐APC. All groups were incubated at room temperature for 30 min. If the sample was peripheral blood, a treatment step was added for incubation with 1 mL red blood cell lysate (BD Biosciences) for 10 min. Then, the samples were resuspended in 1 mL phosphate‐buffered saline (PBS) and concentrated at 250 g for 5 min. Finally, the pellet was resuspended in 300 μL PBS and detected using BD FACS AriaTM II. All flow cytometry antibodies were purchased from Thermo Fisher Scientific Inc. The antibodies information were showed in Table 2.

Adults (≥18 years of age) with chronic HF (functional class II, III, or IV), a left ventricular ejection fraction (EF) of 45% or less, and BNP > 300 ng/L were eligible to participate in the study. Exclusion criteria were as follows: (1) acute renal insufficiency or chronic kidney disease stages III–IV; (2) hepatic insufficiency; (3) pregnant or lactating women; (4) patients with rheumatic immune system disease, severe pneumonia; and (5) malignant tumors (receiving active treatment) or other life-threatening diseases. Blood samples from patients with HF (n = 10) and controls (n = 10) were collected immediately after diagnosis. No significant difference was found in baseline demographics and clinical characteristics between patients with HF and controls (Table 1), indicating no selection bias (p ≥ 0.05). The cell surface antigens were stained according to the standard flow cytometry staining procedures using antibodies specific to CD4, CD196, and CD183 cells (BD Sciences, San Jose, CA, USA). The cells were then treated with red blood cell lysate (BD Sciences, San Jose, CA, USA) and washed twice with phosphate-buffered saline. Flow cytometry was performed using a BD LSR II flow cytometer and analyzed using the FlowJo v7 software (TreeStar, San Carlos, CA, USA).

To generate single-cell suspensions, the tumors and spleen tissues were cut into small pieces, mechanically disrupted, and filtered through a 70-μm strainer. Subsequently, the filtered single cell suspension was mixed with red blood cell lysate (BD Biosciences) to lyse the intermingled red blood cell. Finally, single cell suspensions were combined with fluorescein-labeled antibodies for 1 h at room temperature. The centrifuged single cells were resuspended in PBS and analysed using a flow cytometer (BD Biosciences).

The whole blood samples of 30 patients with PTB and 30 controls were collected. Cell surface markers were then stained with antibodies against CD4 FITC (Cat 555346), CD196 PE (Cat 559562), CD183 ECD (Cat 551128) from BD (San Diego, USA), according to standard procedures. The gating strategy was CD4 + CD183 + CD196- for Th1, CD4 + CD183− CD196 for Th2, and CD4 + CD183-CD196 + for Th17^{26 (link)}. The cells were treated with red blood cell lysate (BD, California, USA) and washed with PBS twice. Flow cytometry of cells was performed with a BD LSRII flow cytometer and analyzed with FlowJo software (v7).

Peripheral blood samples were surface-labeled with anti-CD19 FITC (BD, California, USA), anti-CD3 PC5.5 (BD, California, USA), anti-CD8-PE antibody (BD, California, USA), or anti-CD45 PC7 (BD, California, USA) for 10 min at room temperature. The red blood cells in the blood were lysed with red blood cell lysate (BD, California, USA), then washed with PBS twice, and detected on the Dxflex Flow cytometry (Beckman, California USA). The results were analyzed using the Kaluza v2.1.1 software.

Regarding the protocol for the in vitro mouse Tfh polarization assay, 24‐well round‐bottom tissue culture plates were incubated with 4 μg/mL anti‐CD3 antibody (BioLegend) in PBS at 4°C overnight. Single‐cell suspensions from mouse spleens were prepared, and the red blood cells were lysed with red blood cell lysate (BD). 5 × 10⁵ isolated splenocytes were transferred per well onto the precoated 24‐well round‐bottom tissue culture plates and additionally activated with 1 μg/mL anti‐CD28 (BioLegend, USA), 10 μg/mL anti‐IFN‐γ (BioLegend), 10 μg/mL anti‐IL‐4 antibodies (BioLegend, USA), 20 μg/mL anti‐TGF β (BioLegend), and 10 ng/mL each IL‐6 and IL‐21 (PeproTech) cytokines for 3 days. The percentage of in vitro‐polarized Tfh cells was analyzed using flow cytometry. For the induction of human Tfh cells in vitro, 24‐well plates were precoated with 4 μg/mL anti‐CD3 antibody (OKT3) (BioLegend) at 4°C overnight. The cells were then incubated on the precoated plates in presence of 1 μg/mL anti‐CD28 antibody (BioLegend), 20 μg/mL recombinant human IL‐21 (PeproTech), 5 μg/mL recombinant human IL‐12 (PeproTech), 1 μg/mL recombinant human TGF‐β (PeproTech), and 20 μg/mL recombinant human IL‐6 (PeproTech) at 37°C under 5% CO₂ conditions for 3 days. Plated cells were treated with 0.1 mΜ tapinarof or 50 µM Colivelin TFA as indicated.