Targeting Cardiac Fibrosis with Engineered T cells

Component: Proof-of-Concept Model; Description: Postnᴹᶜᴹ (tamoxifen-inducible Cre in activated cardiac fibroblasts) × RosaOVA (Cre-dependent OVA expression); OT-I CD8⁺ T cells (OVA-specific TCR)

Component: Therapeutic CAR; Description: FAP CAR T cells — scFv from mouse FAP monoclonal antibody (clone 73.3) + mouse CD3ζ + CD28 signaling domains + RIAD peptide (resistance to adenosine/PGE2 suppression)

Component: Control T Cells; Description: MigR1-GFP T cells (control vector); FAP-GFP-CAR T cells (tracking)

Component: T Cell Isolation; Description: Murine splenocytes; CD8⁺ T cell isolation (Miltenyi); OT-I from transgenic mice; retroviral transduction (Phoenix packaging cells)

Component: T Cell Dose; Description: 10⁷ CAR T cells/mouse (FAP CAR); 5×10⁶ CD8⁺ OT-I T cells/mouse (OVA model)

Component: Injury Models; Description: • AngII/PE: 1.5 μg/g/day AngII + 50 μg/g/day PE via 28-day osmotic minipump<br>• MI: Permanent LAD ligation<br>• TAC: Transverse aortic constriction (~50-70% stenosis)<br>• DMD: mdx/mTRᴷᴼ mice

Component: Mouse Strains; Description: C57BL/6; Postnᴹᶜᴹ; RosaOVA; OT-I; mdx/mTRᴷᴼ

Component: Human Samples; Description: Non-failing donor hearts (n=122); HCM (n=27); DCM (n=89) — LV tissue

Parameter: OVA Proof-of-Concept Study; Details: AngII/PE (28 days) + tamoxifen (alternating days); OT-I T cells at week 1 (post-fibrosis establishment); sacrifice at week 4; fibrosis quantitation

Parameter: FAP CAR T Cell Study; Details: AngII/PE (28 days); FAP CAR T cells at weeks 1 and 2; sacrifice at week 4 (primary) and week 8 (persistence); echocardiography (blinded)

Parameter: Human Gene Expression; Details: RNA-seq of LV tissue (238 samples); differential expression (limma package); fibroblast-specific gene signatures

Parameter: Safety Assessments; Details: • Cytokine profiling: 25-plex (days 10, 14, 28, 84)<br>• Cardiotoxicity array: 84 genes (weeks 4, 8)<br>• Immune/inflammatory array: 1659 genes (week 4)<br>• IHC: CD3, CD4, CD8, CD19, F4/80, MPO<br>• Wound healing: 4-mm full-thickness excisional wounds (day 0, 3); closure quantified<br>• Serum amylase: pancreatic toxicity

Parameter: Controls; Details: Saline (no injury); AngII/PE only (no T cells); control T cells (MigR1-GFP); no tamoxifen

Parameter: Sample Sizes; Details: • OVA model: n=6-10 per group<br>• FAP CAR T cell model: n=7-12 per group<br>• Human RNA-seq: n=122, 27, 89

Parameter: Statistical Tests; Details: Student's/Welch's t-test; one-way ANOVA with Tukey's post-hoc; Welch's t-test (cardiotoxicity array)

Analysis Category: Fibrosis Quantification; Methods: Picro-Sirius Red staining; Masson's trichrome; color deconvolution (ImageScope, Aperio); ≥8 sections/mouse; % fibrosis

Analysis Category: Cardiac Function; Methods: Echocardiography (VisualSonics MS400, Vevo 2100); blinded analysis; FS, EDV, ESV, E/E', etc.

Analysis Category: Immunohistochemistry; Methods: FAP (ab207178), GFP (ab6673), CD3 (MCA1477T), CD4 (183685), CD8 (#98941), CD19 (#90176), F4/80 (#70076), MPO (A0398), Vimentin (#9854), cardiac Troponin T (MS-295)

Analysis Category: Gene Expression (Human); Methods: RNA-seq (Illumina truSeq + Nugen Ovation); STAR aligner; edgeR TMM normalization; limma VOOM; differential expression

Analysis Category: Cytokine Profiling; Methods: Luminex 25-plex (MAGPIX); serum from cardiac puncture/IVC

Analysis Category: Cardiotoxicity Array; Methods: RT² Profiler PCR Array (Qiagen PAMM095Z); FFPE RNA; RNeasy FFPE kit; StepOnePlus; normalized to Gapdh

Analysis Category: Immune/Inflammation Array; Methods: HTG ImmunoOncology assay (1659 genes); DESeq2 normalization; Benjamini-Hochberg FDR correction

Analysis Category: Wound Healing; Methods: 4-mm biopsy punches; IVIS imaging; ImageJ quantification; H&E

Analysis Category: T Cell Tracking; Methods: FAP-GFP CAR T cells; IHC co-localization with Fap

Parameter: Cardiac fibrosis (week 4); OVA;Postnᴹᶜᴹ + Tam + OT-I: Significantly reduced; Controls: Widespread fibrosis; Significance: p = 0.0492

Parameter: Heart weight/body weight ratio; OVA;Postnᴹᶜᴹ + Tam + OT-I: Partially rescued; Controls: Increased; Significance: p < 0.0001

Gene: FAP; HCM Fold Change: 3.92; HCM P-value: 6.97×10⁻²⁸; DCM Fold Change: 3.11; DCM P-value: 3.12×10⁻³⁶

Gene: THY1; HCM Fold Change: 3.92; HCM P-value: 6.40×10⁻²⁰; DCM Fold Change: 3.12; DCM P-value: 5.07×10⁻²⁷

Gene: POSTN; HCM Fold Change: 2.64; HCM P-value: 1.40×10⁻¹¹; DCM Fold Change: 2.02; DCM P-value: 3.73×10⁻¹³

Gene: COL1A1; HCM Fold Change: 2.22; HCM P-value: 1.83×10⁻¹⁵; DCM Fold Change: 2.71; DCM P-value: 1.04×10⁻⁴¹

Gene: VIM; HCM Fold Change: 1.26; HCM P-value: 1.43×10⁻¹²; DCM Fold Change: 1.20; DCM P-value: 1.60×10⁻¹⁶

Parameter: Cardiac fibrosis (%); Saline: Low; AngII/PE: High (widespread); AngII/PE + FAP CAR: Significantly reduced; Significance: p < 0.0001

Parameter: Fibrosis reduction; Saline: -; AngII/PE: -; AngII/PE + FAP CAR: Nearly eliminated (5/7 mice); Significance: -

Parameter: Perivascular fibrosis; Saline: -; AngII/PE: Present; AngII/PE + FAP CAR: Persisted (FAP⁻ fibroblasts); Significance: -

Parameter: Ejection fraction; Saline: Normal; AngII/PE: Reduced; AngII/PE + FAP CAR: Partially restored; Significance: p < 0.05

Parameter: E/E' (diastolic function); Saline: Normal; AngII/PE: Impaired; AngII/PE + FAP CAR: Partially restored; Significance: p < 0.05

Parameter: Animal survival (12 weeks); Result: 100% (all survived)

Parameter: Cytokine levels (IL-2, IL-6); Result: AngII/PE increased; FAP CAR did not further increase

Parameter: Cardiotoxicity genes (week 4); Result: Partial changes; resolved by week 8

Parameter: Immune/inflammatory genes (1659); Result: Only 22 genes differentially expressed (minimal inflammation)

Parameter: Immune cell infiltration; Result: CD3⁺, CD4⁺, CD8⁺, MPO⁺: no significant differences; CD19⁺: reduced; F4/80⁺: increased

Parameter: Non-cardiac organ histology; Result: No histological effects detected

Parameter: Wound healing; Result: No delays or defects (FAP CAR vs. control)

Parameter: Serum amylase (pancreatic function); Result: No change

Model: AngII/PE (1-2 weeks); FAP Expression on Activated Fibroblasts: Robust

Model: Myocardial infarction (MI); FAP Expression on Activated Fibroblasts: Present

Model: Transverse aortic constriction (TAC); FAP Expression on Activated Fibroblasts: Present

Model: Muscular dystrophy (mdx/mTRᴷᴼ); FAP Expression on Activated Fibroblasts: Present

Model: Normal/healthy hearts; FAP Expression on Activated Fibroblasts: Undetectable

1. Translation to humans requires further work: The authors explicitly state: "Before this approach can be translated to humans, much work remains to determine if FAP is the optimal target and that safety risks are minimized."

2. FAP expression in other tissues: While FAP is minimally expressed in normal adult tissues, the authors note it is expressed in some fibroblasts in the skin, pancreas, and wound healing sites. Extensive safety analysis was performed, but long-term effects remain unknown.

3. No therapeutic efficacy in a heart failure model with preserved ejection fraction (HFpEF): The study used a pressure-overload model; efficacy in other forms of cardiac fibrosis (e.g., post-MI, diabetic cardiomyopathy) was not fully explored.

4. Mouse CAR T cells have short half-life: The authors note "the short half-life of mouse FAP CAR T cells" necessitating two injections; human FAP CAR T cells may behave differently.

5. Perivascular fibrosis persistence: FAP was not expressed on perivascular fibroblasts, so perivascular fibrosis persisted after treatment—this may limit therapeutic benefit in some contexts.

6. No head-to-head comparison with existing anti-fibrotic therapies: The study does not compare FAP CAR T cells to standard-of-care treatments (e.g., ACE inhibitors, beta-blockers) or other anti-fibrotic agents.

7. Off-target effects potential: The authors acknowledge that "as with any T cell therapy, off-target effects can range from mild to serious" and note that "specific CAR constructs against the same antigen can have vastly different affinities, efficacies, and safety profiles."

8. Single target antigen: FAP is expressed on some normal cells; alternative or combinatorial targeting may be needed for improved safety.

9. No regulatory T cell (Treg) involvement studied: The role of Tregs in modulating the immune response and fibrosis was not investigated.

10. No long-term functional follow-up: While fibrosis was reduced at 4 and 8 weeks, long-term cardiac function (6-12 months) and potential for fibrosis recurrence were not assessed.

11. No human CAR T cell data: All experiments were performed in mouse systems; human FAP CAR T cell safety and efficacy remain to be tested.

12. Potential for on-target/off-tumor effects: FAP is expressed in some cancer-associated fibroblasts and wound healing; repeated dosing or long-term CAR T cell persistence could impair wound healing in future injuries.

Report prepared based on the published Nature article. For full experimental details, supplementary figures, and complete references, please refer to the original publication.