Staining buffer

For cell surface marker staining, draining lymph nodes (dLNs) of each mouse were individually collected and triturated into a single cell suspension. Then the cells were stained with different combinations of flow cytometry antibodies for 30 min at 4°C, which included APC-conjugated anti-mouse CD3 (eBioscience, USA), eFluor450-conjugated anti-mouse CD4 (eBioscience, USA), PE-conjugated anti-mouse CD8a (eBioscience, USA), APC-conjugated anti-mouse B220 (Biolegend, USA), Pacific Blue-conjugated anti-mouse GL-7 (Biolegend, USA), PE-conjugated anti-mouse CD95 (Biolegend, USA), FITC-conjugated anti-mouse CD4 (Biolegend, USA), PE-conjugated anti-mouse PD-1 (Biolegend, USA), and Brilliant Violet 421-conjugated anti-mouse CXCR5 (Biolegend, USA). After washing with staining buffer (eBioscience, USA), the cells were dispersed in 500 μL of staining buffer (eBioscience, USA) and analyzed by flow cytometry (Beckman Coulter, Cytoflex LX).

Mononuclear cells were isolated from the peripheral blood, heart, and spleen as previously described [16 (link)]. Peripheral blood (~200 μL) was collected in EDTA tubes (BD Biosciences). Erythrocytes were lysed with RBC lysis buffer (eBioscience) and the remaining leukocytes were washed with PBS, collected by centrifugation (380g for 10 min at 4°C) and resuspended in 400 μL ice-cold flow cytometry staining buffer (eBiosciences). Splenocytes were isolated by flushing the spleen with 3mL of PBS and then filtering the cells through a 100 μm cell strainer (BD Falcon). Cells were centrifuged at 500g for 5 min at 4°C and resuspended in staining buffer (eBioscience). For cardiac immune cell isolation, whole hearts were flushed with PBS to remove blood and minced into 2mm pieces using a single-edged blade. The tissue was digested in RPMI media containing collagenase-2 (1mg/mL, Worthington), trypsin (1 mg/mL, Invitrogen), and DNase I (10 ug/mL) at 37°C for 45 min with gentle agitation. Cell suspensions were filtered through a 100 μm cell strainer (BD Biosciences) and incubated with 2 mM EDTA in PBS for 5 min at 37°C. Isolates were then centrifuged at 2000g for 20 min on a Ficoll gradient (GE Healthcare), and mononuclear cells were subsequently collected and washed with PBS. Cells were fixed with 1% paraformaldehyde and stored in staining buffer (eBioscience) at 4°C.

Isolation of immune cells from fresh blood was conducted by adding 1 mL ACK lysis buffer per 100 μL of blood, and lysis was performed on ice for 5 min. The lymphocyte fraction was depleted of red blood cells by the lysis. The cells were resuspended in the staining buffer (Thermo Fisher Scientific) and counted, then labeled with the viability stain (Zombie Aqua, BioLegend) in PBS for 15 min at room temperature, washed, and incubated with anti-human CD45-APC-Fire750 (Clone HI30, Biolegend) and anti-human CD3-BV785 (Clone UCHT-1, Biolegend) for 1 h at 4 °C in the staining buffer (Thermo Fisher Scientific). The cells were further washed and fixed in the intracellular (IC) fixation buffer (Thermo Fisher Scientific) overnight at 4 °C. Human CD45⁺ CD3⁺ CAR-T cells were analyzed using the Attune NxT flow cytometer.