Macropinocytosis drives T cell growth by sustaining the activation of mTORC1

Component: Cell Types; Description: • Primary murine splenic CD4⁺ and CD8⁺ T cells (C57BL/6 and mixed backgrounds)<br>• OTII TCR transgenic T cells (specific for Ova peptide 323-329/I-Aᵇ)<br>• Human PBMC-derived CD4⁺ and CD8⁺ T cells<br>• RasGRP1 mutant mouse T cells<br>• Murine bone marrow-derived macrophages (CSF1-stimulated; SEM comparison)

Component: Macropinocytosis Probes; Description: • 70 kDa fluorescein-dextran (Fdex) — large molecular weight, limits uptake by small-scale endocytosis<br>• BSA-Alexa 488 or Alexa 647 — macropinocytosis probe; retained after fixation<br>• DQ Red BSA — fluoresces only upon lysosomal digestion

Component: Pharmacological Inhibitors; Description: • EIPA (5-(N-Ethyl-N-isopropyl)amiloride) — specific macropinocytosis inhibitor (Na⁺/H⁺ exchanger)<br>• Jasplakinolide + Blebbistatin (J/B) — actin cytoskeletal inhibitors<br>• PitStop 2 — clathrin-mediated endocytosis inhibitor (control)<br>• FTS (Farnesyl thiolsalylic acid) — Ras inhibitor<br>• LY294002 — PI3K inhibitor<br>• EHT 1864 — Rac1 inhibitor<br>• IPA-3 — Pak1 inhibitor<br>• Torin 1 — mTORC1 inhibitor<br>• NH₄Cl — lysosomal pH neutralizer (inhibits protein degradation)

Component: Activation Stimuli; Description: Anti-CD3 + anti-CD28 mAb; PHA (human); Ova protein immunization (in vivo)

Component: Mouse Models; Description: • OTII TCR Tg → CD45.1 recipient (in vivo macropinocytosis)<br>• RasGRP1 mutant mice (Ras pathway)

Component: Key Readouts; Description: • Probe uptake (flow cytometry, confocal microscopy)<br>• Cell size (FSC-A, flow cytometry)<br>• mTORC1 activation (phospho-S6, flow cytometry)<br>• NFκB activation (flow cytometry)<br>• Macropinosome morphology (SEM, confocal microscopy)

Parameter: In Vitro Macropinocytosis; Details: T cells stimulated with CD3/28 mAb for 12-24 h; probes added for last 4-8 h; flow cytometry analysis of uptake; inhibitor treatments 15 min before probe addition

Parameter: In Vivo Macropinocytosis; Details: OTII T cells adoptively transferred; recipients immunized with Ova (footpad, RIBI adjuvant); BSA-Alexa 647 injected; draining lymph node harvest; flow cytometry of OTII T cells

Parameter: Human T Cell Studies; Details: PBMC isolated from buffy coats; stimulated with CD3/28 mAb or PHA; BSA-Alexa 488 uptake; flow cytometry

Parameter: Cell Growth Assay; Details: T cells stimulated 12-20 h with CD3/28 mAb; inhibitors added at 12 h; cell size (FSC-A) measured at 20 h; growth calculated relative to no inhibitor

Parameter: mTORC1 Activation; Details: Phospho-S6 staining by flow cytometry; inhibitors added at 12 h; time course 1-8 h post-inhibitor

Parameter: AA Sufficiency Experiment; Details: T cells stimulated 12 h in complete medium; washed; recultured in minimal medium ± 20 AA or L,Q,R,S for 2 h; ± EIPA; phospho-S6 measured

Parameter: Confocal Microscopy; Details: Poly-L-lysine-coated coverslips; probes ± inhibitors; staining: anti-CD4/CD8, LAMP-1, LAMP-2, phalloidin (actin), Hoechst; Leica SP5 confocal

Parameter: Scanning EM; Details: T cells ± CD3/28 stimulation; BM macrophages + CSF1; gold coating; Amray 1900 SEM

Parameter: Replicates; Details: n=3-15 independent experiments; ≥150 cells counted for microscopy

Parameter: Statistical Tests; Details: Student's 1-sample or 2-sample 2-sided t-test; P<0.05, P<0.01, P<0.001, P<0.0001

Analysis Category: Macropinocytosis Measurement; Methods: Flow cytometry: MFI of Fdex/BSA uptake; ratio of stimulated vs. unstimulated; percentage macropinocytosis with inhibitors = [(MFI_inhibitor_37°C - MFI_4°C) / (MFI_no_inhibitor_37°C - MFI_4°C)] × 100

Analysis Category: Cell Growth; Methods: Flow cytometry: median FSC-A of CD4⁺/CD8⁺ T cells; % growth = [(FSC_inhibitor_20h - FSC_12h) / (FSC_no_inhibitor_20h - FSC_12h)] × 100

Analysis Category: mTORC1 Activation; Methods: Intracellular flow cytometry: PE-Cy7-phospho-S6 (Cell Signaling, clone D57.2.2E); methanol permeabilization

Analysis Category: Lysosomal Traffic; Methods: DQ Red BSA fluorescence (flow cytometry); colocalization with LAMP-1/LAMP-2 (confocal microscopy)

Analysis Category: Confocal Microscopy; Methods: Leica SP5; 63× objective; staining: anti-CD4/CD8, anti-LAMP-1/2, phalloidin-Alexa 488, Hoechst

Analysis Category: Scanning EM; Methods: Amray 1900 field emission SEM; gold coating; hexamethyldisilazane dehydration

Analysis Category: Gating Strategy; Methods: FSC-A/SSC-A → single cells (FSC-A/FSC-H) → CD4⁺/CD8⁺ lymphocytes (Supplementary Fig. 8)

Cell Type: Murine CD4⁺ T cells; Probe: 70 kDa Fdex; Stimulation: CD3/28 mAb (24 h); Fold Increase (Stimulated vs. Unstimulated): ~2× (n=10)

Cell Type: Murine CD8⁺ T cells; Probe: 70 kDa Fdex; Stimulation: CD3/28 mAb (24 h); Fold Increase (Stimulated vs. Unstimulated): ~2× (n=10)

Cell Type: Murine CD4⁺ T cells; Probe: BSA-Alexa 488; Stimulation: CD3/28 mAb (20 h); Fold Increase (Stimulated vs. Unstimulated): Significant increase (n=15)

Cell Type: Murine CD8⁺ T cells; Probe: BSA-Alexa 488; Stimulation: CD3/28 mAb (20 h); Fold Increase (Stimulated vs. Unstimulated): Significant increase (n=12)

Cell Type: Human CD4⁺ T cells; Probe: BSA-Alexa 488; Stimulation: CD3/28 mAb (20 h); Fold Increase (Stimulated vs. Unstimulated): Significant (n=3)

Cell Type: Human CD8⁺ T cells; Probe: BSA-Alexa 488; Stimulation: CD3/28 mAb (20 h); Fold Increase (Stimulated vs. Unstimulated): Significant (n=3)

Cell Type: OTII T cells (in vivo); Probe: BSA-Alexa 647; Stimulation: Ova immunization; Fold Increase (Stimulated vs. Unstimulated): Significant increase (P<0.001)

Cell Type: 4°C control; Probe: All probes; Stimulation: -; Fold Increase (Stimulated vs. Unstimulated): No uptake

Observation: Macropinosomes per CD4⁺ T cell (37°C); Result: 1 to ≥10 per cell

Observation: Macropinosomes per CD4⁺ T cell (4°C); Result: None

Observation: LAMP-1 colocalization with DQ Red BSA; Result: 77.3 ± 3.9% (n=35 cells)

Observation: LAMP-2 colocalization with DQ Red BSA; Result: 84.9 ± 3.8% (n=23 cells)

Observation: Actin polymerization structures; Result: Loops extending 1-2 μm from surface

Inhibitor: EIPA; Target: Na⁺/H⁺ exchanger; Effect on Macropinocytosis (CD8⁺ T cells): Significant inhibition (P<0.0001)

Inhibitor: J/B; Target: Actin cytoskeleton; Effect on Macropinocytosis (CD8⁺ T cells): Significant inhibition (P<0.0001)

Inhibitor: PitStop 2; Target: Clathrin-mediated endocytosis; Effect on Macropinocytosis (CD8⁺ T cells): No effect (NS)

Inhibitor: FTS; Target: Ras; Effect on Macropinocytosis (CD8⁺ T cells): No effect (NS)

Inhibitor: LY294002; Target: PI3K; Effect on Macropinocytosis (CD8⁺ T cells): Partial inhibition

Inhibitor: EHT 1864; Target: Rac1; Effect on Macropinocytosis (CD8⁺ T cells): Partial inhibition

Inhibitor: IPA-3; Target: Pak1; Effect on Macropinocytosis (CD8⁺ T cells): Partial inhibition

Inhibitor: RasGRP1 KO; Target: Ras pathway; Effect on Macropinocytosis (CD8⁺ T cells): No effect

Inhibitor: EIPA; % Growth (vs. No Inhibitor): ~10-20%; Significance: P<0.0001

Inhibitor: J/B; % Growth (vs. No Inhibitor): ~20-30%; Significance: P<0.0001

Inhibitor: PitStop 2; % Growth (vs. No Inhibitor): ~90%; Significance: NS

Inhibitor: LY294002; % Growth (vs. No Inhibitor): ~40%; Significance: P<0.01

Inhibitor: EHT 1864; % Growth (vs. No Inhibitor): ~30%; Significance: P<0.01

Inhibitor: IPA-3; % Growth (vs. No Inhibitor): ~40%; Significance: P<0.01

Inhibitor: FTS; % Growth (vs. No Inhibitor): ~100%; Significance: NS

Inhibitor: Torin 1; % Growth (vs. No Inhibitor): ~30%; Significance: P<0.05

Inhibitor: Correlation; % Growth (vs. No Inhibitor): Macropinocytosis inhibition vs. growth inhibition; Significance: Strong correlation (Fig. 4b)

Inhibitor: EIPA; Effect on Phospho-S6: Blocked (as early as 1 h)

Inhibitor: J/B; Effect on Phospho-S6: Blocked

Inhibitor: PitStop 2; Effect on Phospho-S6: No effect

Inhibitor: LY294002; Effect on Phospho-S6: Blocked

Inhibitor: EHT 1864; Effect on Phospho-S6: Blocked

Inhibitor: IPA-3; Effect on Phospho-S6: Blocked

Inhibitor: Torin 1; Effect on Phospho-S6: Blocked

Inhibitor: NH₄Cl; Effect on Phospho-S6: No effect (protein degradation not required)

Media Condition: 20 AA; mTORC1 Activation (vs. 0 AA): Sustained (P<0.01); EIPA Effect: Blocked

Media Condition: L,Q,R,S only; mTORC1 Activation (vs. 0 AA): Sustained (P<0.01); EIPA Effect: Blocked

Media Condition: 0 AA; mTORC1 Activation (vs. 0 AA): Baseline; EIPA Effect: -

Media Condition: L or R omitted (from LQRS); mTORC1 Activation (vs. 0 AA): Not sustained; EIPA Effect: -

Media Condition: S or Q omitted (from LQRS); mTORC1 Activation (vs. 0 AA): Sustained; EIPA Effect: -

1. Pharmacological inhibitor specificity: The authors used several inhibitors with potential off-target effects. They note that "at the concentrations used, inhibitors showed minimal toxicity," but acknowledge the limitations of chemical inhibitors.

2. Incomplete mechanistic understanding: The authors state: "Why T cells are dependent upon macropinocytosis for sustained mTORC1 activation and growth even under conditions where AA are freely available in the extracellular space remains to be determined." The exact mechanism remains speculative.

3. Lysosomal AA transport unresolved: The authors propose that "a requirement for macropinocytosis for sustained mTORC1 activation in T cells could be explained by an absence of relevant AA transporters at the lysosomal membrane," but this was not directly demonstrated.

4. Correlative evidence for mTORC1-growth link: While the study shows correlation between macropinocytosis, mTORC1 activation, and growth, the direct causal relationship beyond inhibitor studies was not fully established.

5. In vitro primary cell studies only: All experiments were performed on primary T cells in culture; physiological relevance in vivo was only partially addressed with the OTII immunization model for probe uptake, not for growth or mTORC1.

6. Single cell type focus: While the authors suggest macropinocytosis may be "applicable to the growth of other primary cell types," this was not demonstrated in the study.

7. No assessment of downstream consequences: The study did not examine whether blocking macropinocytosis affects T cell effector functions (e.g., cytokine production, proliferation beyond G1, differentiation).

8. Potential confounding of EIPA effects: EIPA inhibits Na⁺/H⁺ exchange, which could affect pH regulation beyond macropinocytosis, potentially influencing mTORC1 indirectly.

9. LQRS amino acid specificity: While L and R were identified as critical, the study did not exhaustively test all possible AA combinations or determine the precise mechanistic roles of each AA.

10. Human T cell studies limited: Human T cell macropinocytosis was demonstrated but mechanistic studies (inhibitors, mTORC1, growth) were primarily performed in murine cells.

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