Fig 2: Comparison of age [years] (A) and BMI [kg/m2] (B) between control cohort (CTRL, n = 10) and baseline cohort with morbid obesity (Baseline, n = 38) and comparison of abundance of PBMCs [% singlets] including CD3+ T cells (CD3+), CD4+ T cells (CD3+, CD56-, CD4+), CD8+ T cells (CD3+, CD56-, CD8+), B cells (CD19+), NK cells (Lin-, CD56+), HLA-DR- CD14+, Monocytes (HLA-DR+, CD14+, CD16var) divided in non-classical monocytes (NCM)(CD14low, CD16high), intermediate monocytes (IM) (CD14high, CD16high) and classical monocytes (CM) (CD14high, CD16low) (C–L). Statistical analysis was performed using a two-sided unpaired t-test (Age, CD3+ T cells, CD4+ T cells, CD8+ T cells, NCM, IM and CM), a Welch’s test (BMI, B cells) or a Mann-Whitney test (HLA-DR- CD14+, Monocytes, NK cells) with α = 0.05. *p-value ≤ 0.05, ***p-value ≤ 0.001, ****p-value ≤ 0.0001.

Fig 3: (A) Overview of the experimental plan comparing T cell culture with BECA-S and BECA-Auto. (B) Cell population (solid line) and cell viability (dashed line) in BECA-S and BECA-Auto. Inverted triangle indicates expansion of culture for BECA-S (black) and BECA-Auto (green). (C) Cell population in BECA-S and BECA-Auto. (D) Left: Percentage population of CD3 + cells in culture as analysed by flow cytometry on Day 0, Day 7, and Day 10 for each donor. Level of 80% live lymphocytes is indicated by red dotted line. Right: Mean and standard deviation of values shown in Left. (E) Percentage population of CD4 and CD8 T cells in culture as analysed by flow cytometry on Day 0, Day 7, and Day 10. (F) Ratio of CD4/CD8 T cells as calculated from values in 3E. (G) Heatmap of percentage population of memory T cells in culture as analysed by flow cytometry on Day 0, Day 7, and Day 10 for each donor. TN: CCR7 + CD45RA+, TCM: CCR7 + CD45RA-, TEM: CCR7-CD45RA-, TEMRA: CCR7-CD45RA+. (H) Percentage population of TIM3 + PD1 + exhausted T cells. P-values indicated were calculated using paired t-test. n.s. not significant, * P < 0.05.

Fig 4: CSPG4.CAR-T generation and characterization. (A) Flow cytometry histogram shows CSPG4.CAR expression in CSPG4.CAR-Ts lymphocytes (pink line) compared to NTs cells (cyan line). (B) Immunofluorescence staining for CSPG4.CAR (red) and CD3 (green) markers. Nuclei were counterstained with DAPI (blue). Scale bars represent 100 μm. (C) Representative dot plots of the cytotoxic activity of CSPG4.CAR-T compared to CD3-NTs lymphocytes. Flow cytometry plots show T cells (effector on the x-axis) and U87-MG (target on the y-axis) on day 4 of co-culture. Anti-CD3 and anti-CSPG4 conjugated antibodies were used as markers for T cells and U87-MG, respectively. (D) Representative image of the in vivo treatment created with BioRender. (E) Flow cytometry analysis showing the presence of CSPG4.CAR-T lymphocytes in the spleen compared to native mouse lymphocytes, identified by CD45RA (human) and CD45RC (mouse) markers, 30 days post treatment.

Fig 5: Histological analysis of cardiac inflammation. (A) Representative confocal images of WT and mdx mice’ hearts treated with CD3-NTs or CSPG4.CAR-T cells and stained for CSPG4 in green, and F4/80 in red. Nuclei are counterstained with DAPI in blue. Scale bar 50 μm. (B) Charts indicate positive area expressed as percentage of CSPG4 and F4/80 on whole area. N = 3 section per N = 3 biological replicates. (C) Western blot analysis of CSPG4 in total hearts of WT and mdx mice. Bar chart shows optical density (OD) of protein bands normalized to Vinculin (VCL). N = 3 biological replicates per group were analyzed. Error bars show SEM. The repeated measure ANOVA with Tukey correction was used to evaluate the differences between means, p: * p < 0.05, *** p < 0.001, **** p < 0.0001.