Biomimetic Tolerogenic Artificial Antigen Presenting Cells for Regulatory T Cell Induction

Component: Core Material; Description: • PLGA-only: Poly(lactic-co-glycolic acid), acid-terminated, 50:50 lactate:glycolide, MW 34,000-58,000 Da<br>• PLGA/PBAE blend: 75:25 w/w blend of PLGA and PBAE

Component: PBAE Synthesis; Description: Two-step Michael addition: (1) 4,4'-trimethylenedipiperidine + 1,4-butanediol diacrylate (1.2:1) at 90°C, 24 h; (2) end-capping with 1-(3-aminopropyl)-4-methylpiperazine; Mn ~28 kDa, Mw ~137 kDa

Component: Particle Size; Description: ~3 μm diameter (similar for PLGA and PLGA/PBAE)

Component: Zeta Potential; Description: PLGA: -2.47 ± 2.84 mV; PLGA/PBAE: -2.77 ± 2.61 mV

Component: Signals Presented; Description: • Signal 1 (extracellular): Anti-CD3 antibody (clone 145-2C11 for mouse; OTK3 for human)<br>• Signal 2 (extracellular): Anti-CD28 antibody<br>• Signal 3 (soluble, released): TGF-β (recombinant mouse, encapsulated in particle core)

Component: Conjugation Method; Description: EDC/NHS chemistry (couples primary amines on proteins to carboxylic acid-terminated PLGA)

Component: Protein Doses Tested; Description: 1/5× (0.8 μg anti-CD3 / 1 μg anti-CD28 per mg particles), 1× (4 μg/5 μg), 5× (20 μg/25 μg)

Component: TGF-β Loading; Description: 2.5 μg TGF-β + 1 mg BSA per 50 mg polymer; encapsulated via probe sonication + double emulsion

Component: Release Profile; Description: Sustained release over 1 month; total release: PLGA 1.5 ng/mg, PLGA/PBAE 0.26 ng/mg

Component: Protein Retention (7 days); Description: PLGA: 67%; PLGA/PBAE: 86%

Parameter: Cell Sources; Details: • Murine: C57BL/6J mice (male, 8-12 weeks); CD4+CD25- naïve T cells isolated from splenocytes via magnetic bead depletion (Miltenyi); Thy1.1 reporter mice for suppression assays<br>• Human: Naïve CD4+ T cells from peripheral blood (Supplementary Figure 2)

Parameter: In Vitro iTreg Induction; Details: 5-day culture with aAPCs (0.001-1 mg/mL), TGF-β (5 ng/mL), IL-2 (400 U/mL); Foxp3 expression measured by flow cytometry

Parameter: Suppression Assay; Details: CFSE-labeled Thy1.1+ responder cells + iTreg suppressors at ratios 1:1, 1:2, 1:4, 1:8, 0:1; stimulated with anti-CD3/CD28 Dynabeads (1:2 bead:responder); proliferation measured by CFSE dilution at day 3

Parameter: In Vivo Study; Details: Single intravenous (retroorbital) injection of TGF-β-loaded TolAPCs (2 mg) into C57BL/6J mice; spleens and lymph nodes harvested at day 5; CD4+Foxp3+ cells quantified by flow cytometry

Parameter: Controls; Details: • No treatment (untreated mice)<br>• TGF-β-loaded unconjugated PLGA/PBAE particles (no anti-CD3/CD28)<br>• Cytokines only (no aAPCs)<br>• Anti-human CD3 aAPCs (non-binding control)

Parameter: Replicates; Details: Technical replicates: n=3-8; In vivo: n=5 animals per condition

Parameter: Statistical Tests; Details: One-way/two-way ANOVA with Tukey's or Sidak's post-tests; two-tailed t-test; Mann-Whitney test (Figure 1)

Analysis Category: Particle Characterization; Methods: • SEM (LEO FESEM) with ImageJ size analysis<br>• Zeta potential (ZetaSizer, Malvern)<br>• GPC for PBAE molecular weight (Ultrastrygel column, THF/DMSO/piperidine mobile phase)

Analysis Category: Protein Quantification; Methods: Fluorescently labeled anti-CD3/CD28; fluorescence intensity measured after conjugation; stability assessed over 7 days at 37°C in PBS

Analysis Category: TGF-β Release; Methods: Sandwich ELISA (Biolegend) of supernatants from particles incubated in PBS at 37°C over 1 month

Analysis Category: aAPC-T Cell Binding; Methods: • DiD-labeled particles + CFSE-labeled T cells; 1 h incubation at 37°C<br>• Confocal microscopy (Zeiss 710 LSM)<br>• Flow cytometry (Accuri C6): CFSE+DiD+ double-positive events; DiD MFI of CFSE+ cells

Analysis Category: iTreg Phenotype; Methods: Flow cytometry: CD4, CD25, Foxp3 (clone FJK-16s), CD39, CD73; anti-mouse antibodies from Biolegend/eBioscience

Analysis Category: Cytokine Profiling; Methods: Proteome Profiler Mouse Cytokine Array Kit (R&D Systems); ImageJ pixel density analysis

Analysis Category: Foxp3 Methylation; Methods: Pyrosequencing (EpigenDx) of CpG motifs within foxp3 locus (TSDR)

Analysis Category: iTreg Stability; Methods: Culture in IL-2 (40 U/mL) for 7 days; Foxp3 expression measured at days 2 and 7

Analysis Category: Suppression Assay; Methods: CFSE dilution; Division Index (DI) calculated in FlowJo; % Suppression = 100 − (DI_sample/DI_control) × 100

Parameter: Anti-CD3 conjugation (1× dose); PLGA aAPC: Lower; PLGA/PBAE aAPC: Significantly higher (all doses); Significance: p < 0.01 to p < 0.0001

Parameter: Anti-CD28 conjugation (1× dose); PLGA aAPC: Lower; PLGA/PBAE aAPC: Significantly higher (all doses); Significance: p < 0.01 to p < 0.0001

Parameter: Protein retention (7 days, 37°C); PLGA aAPC: 67%; PLGA/PBAE aAPC: 86%; Significance: p < 0.01

Parameter: TGF-β release (total); PLGA aAPC: 1.5 ng/mg; PLGA/PBAE aAPC: 0.26 ng/mg; Significance: -

Parameter: % T cells bound (0.05 mg dose); PLGA aAPC: 8 ± 2%; PLGA/PBAE aAPC: 30 ± 2%; p-value: p < 0.0001

Parameter: % T cells bound (0.01 mg dose); PLGA aAPC: ~5%; PLGA/PBAE aAPC: ~18%; p-value: p < 0.0001

Parameter: MFI of binding events (0.05 mg); PLGA aAPC: Baseline; PLGA/PBAE aAPC: 4.5× higher; p-value: p < 0.0001

Parameter: Specificity for CD3+ cells; PLGA aAPC: Modest; PLGA/PBAE aAPC: Significantly higher; p-value: -

Condition: PLGA aAPC + TGF-β; % Foxp3+ (1× protein, 0.1 mg/mL): ~13% CD25+Foxp3+

Condition: PLGA/PBAE aAPC + TGF-β; % Foxp3+ (1× protein, 0.1 mg/mL): >70% CD25+Foxp3+

Condition: PLGA/PBAE aAPC (no TGF-β); % Foxp3+ (1× protein, 0.1 mg/mL): ~3% Foxp3+ (CD25+ only)

Condition: Cytokines only (no aAPC); % Foxp3+ (1× protein, 0.1 mg/mL): Very low

Marker: CD25+Foxp3+; PLGA aAPC-iTreg: ~13%; PLGA/PBAE aAPC-iTreg: >70%

Marker: CD39+CD73+ co-expression; PLGA aAPC-iTreg: Lower; PLGA/PBAE aAPC-iTreg: Highest

Marker: Foxp3 TSDR methylation; PLGA aAPC-iTreg: Partially demethylated; PLGA/PBAE aAPC-iTreg: Partially demethylated

Marker: Stability (Foxp3+ at day 7); PLGA aAPC-iTreg: ~20% remaining; PLGA/PBAE aAPC-iTreg: ~20% remaining

Suppressor:Responder Ratio: 1:1; PLGA aAPC-iTreg: Moderate suppression; PLGA/PBAE aAPC-iTreg: Almost complete suppression; Significance: p < 0.0001

Suppressor:Responder Ratio: 1:2; PLGA aAPC-iTreg: Partial suppression; PLGA/PBAE aAPC-iTreg: Strong suppression; Significance: p < 0.001

Suppressor:Responder Ratio: 1:4; PLGA aAPC-iTreg: Minimal suppression; PLGA/PBAE aAPC-iTreg: Moderate suppression; Significance: p < 0.01

Tissue: Spleen (% Foxp3+ of CD4+); Untreated: Baseline; PLGA TolAPC: No significant change; PLGA/PBAE TolAPC: 14.8 ± 2.4%; Effect: Trend (not significant)

Tissue: Lymph nodes (% Foxp3+ of CD4+); Untreated: ~11.8%; PLGA TolAPC: ~13.0%; PLGA/PBAE TolAPC: 14.1 ± 1.1%; Effect: ~20% relative increase (p < 0.05)

1. Modest in vivo effect: The ~20% relative increase in Foxp3+ cells in lymph nodes, while statistically significant, represents a modest absolute increase (~2.3 percentage points). The authors note this was a "single dose" study and effects were "modest."

2. Data availability restricted: "The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study."

3. No antigen specificity: The aAPCs use anti-CD3/CD28 antibodies for polyclonal T cell stimulation, not antigen-specific MHC-peptide complexes. This limits their ability to induce antigen-specific tolerance.

4. Stability of iTregs: The authors acknowledge that "iTregs are relatively unstable in vitro" with only ~20% Foxp3+ cells remaining after 7 days, stating "stability of iTregs is an important challenge that should be addressed to maximize the therapeutic potential of these cells."

5. No therapeutic disease model: While in vivo Treg induction was demonstrated, no autoimmune disease model (e.g., EAE, T1D) or transplant model was tested to show therapeutic benefit.

6. TGF-β loading efficiency lower in PLGA/PBAE: PLGA/PBAE particles released less total TGF-β (0.26 vs. 1.5 ng/mg) than PLGA-only particles, yet still outperformed PLGA in iTreg induction—suggesting the enhanced protein presentation/binding is the dominant factor, but the lower loading efficiency could be a limitation for translation.

7. Human data preliminary: Human iTreg induction was only shown in Supplementary Figure 2 with relatively similar PLGA and PLGA/PBAE performance at higher doses; the authors state "future studies should optimize aAPCs and TolAPCs for human iTreg induction."

8. No in vivo tracking/biodistribution: Particle half-life (cited 11.6 min from prior work) and biodistribution were not measured in this study.

9. Non-specific protein adsorption: The authors suggest PBAE "may increase nonspecific adsorption of protein to the polymer surface," which could lead to uncontrolled presentation of multiple proteins or serum protein corona formation in vivo.

10. No comparison to other Treg induction methods: The TolAPCs were not benchmarked against other established Treg induction strategies (e.g., TGF-β + retinoic acid, rapamycin-treated DCs, or other nanoparticle systems).

11. Polyclonal vs. antigen-specific suppression: The suppression assays used polyclonal stimulation with anti-CD3/CD28 beads; the ability of these iTregs to suppress antigen-specific responses was not demonstrated.

12. PBAE degradation products: While PBAE is biodegradable, the immunogenicity or toxicity of its degradation products was not characterized.

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