Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes

Component: Genetic Model; Description: Conditional dynamin 2 (Dnm2) knockout mice: Cd4-creDnm2^(flox/flox) ("Dnm2 KO") — dynamin 2 ablated specifically in T cells; Cd4-creDnm2^(flox/+) ("Dnm2 HET") used as controls

Component: Dynamin 2 Function; Description: Essential GTPase for both clathrin-dependent and clathrin-independent endocytosis; the only dynamin isoform expressed in T lymphocytes

Component: Key Proteins Studied; Description: TCR (surface expression, signaling), CD5 (TCR signal strength reporter), CD25 (IL-2Rα), CD69 (activation marker), mTORC1 (S6, S6K1, 4E-BP1), c-Myc, Glut1, LC3B (autophagy marker)

Component: Metabolic Readouts; Description: Extracellular acidification rate (ECAR) — glycolysis; Oxygen consumption rate (OCR) — oxidative phosphorylation; Glucose uptake (2-NBDG)

Component: In Vivo Models; Description: • Rag1 KO mice (lymphopenic) — homeostatic proliferation<br>• LM-OVA infection — antigen-specific CD8 T cell expansion (OT-I TCR transgenic)<br>• Mixed bone marrow chimeras — cell-intrinsic analysis

Component: In Vitro Stimulation; Description: Plate-bound anti-CD3 + soluble anti-CD28 antibodies (TCR crosslinking + costimulation)

Component: Inhibitors; Description: Bafilomycin A1 (lysosomal inhibitor, autophagy flux measurement)

Parameter: TCR Internalization; Details: Flow cytometry: surface TCRβ staining at 0, 2, 4, 8, 24 h after anti-CD3 stimulation

Parameter: TCR Signal Strength; Details: CD5 surface expression (correlates with TCR signal strength in vivo) measured by flow cytometry in thymocytes and peripheral T cells

Parameter: Control for Thymic Egress; Details: FTY720 treatment of wild-type mice to block S1P1-mediated egress; CD5 expression measured in mature thymocytes

Parameter: In Vivo Homeostatic Proliferation; Details: CFSE-labeled naive T cells (1:1 HET:KO) cotransferred into Rag1 KO recipients; CFSE dilution (division) and cell recovery at day 4-14

Parameter: In Vivo Antigen-Specific Proliferation; Details: OT-I GzmB-creDnm2^(flox/flox) T cells (KO) vs. OT-I Dnm2^(flox/flox) (WT) cotransferred 1:1 into B6 recipients; LM-OVA infection; blood T cell counts over time

Parameter: In Vitro Proliferation; Details: ³H-thymidine incorporation (DNA synthesis); cell size by FSC/SSC; survival by 7-AAD/annexin V

Parameter: Signaling Analysis; Details: Western blot (phospho-ERK, phospho-JNK, phospho-p38, phospho-Lck, phospho-ZAP70, phospho-S6K1, phospho-4E-BP1, c-Myc, Glut1, LC3B); intracellular flow cytometry (phospho-S6, phospho-4E-BP1)

Parameter: Metabolic Studies; Details: Seahorse XF96 analyzer (ECAR, OCR); 2-NBDG glucose uptake (flow cytometry); qRT-PCR for Myc, Hk2, Pkm, Ldha, Ppat, Got1

Parameter: Autophagy Measurement; Details: LC3B Western blot ± bafilomycin A1 (4 h treatment); LC3B II quantification by densitometry

Parameter: Sample Sizes; Details: n=3-9 per group; data combined from 2-5 experiments

Parameter: Statistical Tests; Details: Unpaired Student's t-test (two groups); one-way ANOVA with Tukey post-test (multigroup comparisons); P < 0.05 considered significant

Analysis Category: TCR Internalization; Methods: Flow cytometry: surface TCRβ staining (anti-TCRβ antibody); time course after anti-CD3 stimulation

Analysis Category: TCR Signal Strength; Methods: Flow cytometry: CD5 surface expression on thymocytes (DP, SP) and peripheral naive T cells

Analysis Category: Cell Proliferation; Methods: • In vivo: CFSE dilution (flow cytometry); cell counts (absolute number recovered)<br>• In vitro: ³H-thymidine incorporation (cpm)

Analysis Category: Cell Survival; Methods: 7-AAD and annexin V staining (flow cytometry)

Analysis Category: Phospho-Protein Analysis; Methods: • Western blot: phospho-ERK, phospho-JNK, phospho-p38, phospho-Lck, phospho-ZAP70, phospho-S6K1, phospho-Tsc2, phospho-Raptor, phospho-AMPKα<br>• Intracellular flow cytometry: phospho-S6, phospho-4E-BP1, phospho-Akt (T308, S473)

Analysis Category: Protein Expression; Methods: Western blot: c-Myc, Glut1, LC3B (I and II isoforms), S6K1 (loading control), actin (loading control)

Analysis Category: Gene Expression; Methods: qRT-PCR: Myc, Hk2, Pkm, Ldha, Ppat, Got1; normalized to Hprt

Analysis Category: Metabolic Flux; Methods: Seahorse XF96: ECAR (glycolysis), OCR (oxidative phosphorylation)

Analysis Category: Glucose Uptake; Methods: 2-NBDG fluorescent glucose analog; flow cytometry

Analysis Category: Autophagy Flux; Methods: LC3B II accumulation ± bafilomycin A1 (lysosomal inhibitor); densitometry quantification

Analysis Category: Cell Size/Growth; Methods: Flow cytometry: forward scatter (FSC) and side scatter (SSC)

Parameter: TCR down-modulation (8 h post-stimulation); Dnm2 HET: ~50% reduction; Dnm2 KO: Largely abolished; Significance: -

Parameter: CD5 expression (mature thymocytes/peripheral T cells); Dnm2 HET: Normal; Dnm2 KO: Markedly reduced; Significance: -

Parameter: CD5 expression (double-positive thymocytes); Dnm2 HET: Normal; Dnm2 KO: Normal (no difference); Significance: ns

Assay: Homeostatic proliferation (Rag1 KO, CD8, day 8); Dnm2 HET: High recovery; Dnm2 KO: >100-fold fewer cells; Fold Difference: -

Assay: CFSE dilution (homeostatic proliferation, day 4); Dnm2 HET: Multiple divisions; Dnm2 KO: Fewer divisions; Fold Difference: -

Assay: Homeostatic proliferation (CD4, day 14); Dnm2 HET: Normal expansion; Dnm2 KO: Defective expansion; Fold Difference: -

Assay: Antigen-specific expansion (LM-OVA, OT-I, day 7); Dnm2 HET: ~5×10⁵ cells/mL blood; Dnm2 KO: ~1×10⁵ cells/mL blood; Fold Difference: ~5-fold reduction

Parameter: ³H-thymidine incorporation (72 h); Dnm2 HET: Robust; Dnm2 KO: ~90% reduction; Significance: P < 0.001

Parameter: CD69 up-regulation (early); Dnm2 HET: Yes; Dnm2 KO: Yes (normal initial activation); Significance: ns

Parameter: CD25 expression (CD4, 20 h); Dnm2 HET: High; Dnm2 KO: Reduced; Significance: P < 0.05

Parameter: Cell size/blast formation (48 h); Dnm2 HET: Large blasts; Dnm2 KO: Smaller, no blasts; Significance: -

Parameter: Survival (24-48 h); Dnm2 HET: High; Dnm2 KO: Normal (no excessive death); Significance: ns

Parameter: Phospho-S6 (mTORC1); Time Point: 8 h; Dnm2 HET: High; Dnm2 KO: High (similar); Significance: ns

Parameter: Phospho-S6 (mTORC1); Time Point: 20 h; Dnm2 HET: Sustained; Dnm2 KO: Lost; Significance: P < 0.05

Parameter: Phospho-S6K1 (20 h); Time Point: 20 h; Dnm2 HET: Robust; Dnm2 KO: Reduced; Significance: -

Parameter: Phospho-4E-BP1 (20 h); Time Point: 20 h; Dnm2 HET: Sustained; Dnm2 KO: Reduced; Significance: P < 0.05

Parameter: LC3B II (autophagy); Time Point: 48 h; Dnm2 HET: Basal; Dnm2 KO: Increased; Significance: -

Parameter: LC3B II + bafilomycin A1; Time Point: 48 h; Dnm2 HET: Increased; Dnm2 KO: Further increased (autophagic flux); Significance: -

Parameter: c-Myc protein (20 h); Dnm2 HET: Robust; Dnm2 KO: Failed to express; Significance: -

Parameter: Myc mRNA (4 h); Dnm2 HET: Peak; Dnm2 KO: Similar; Significance: ns

Parameter: Myc mRNA (8 h); Dnm2 HET: High; Dnm2 KO: ~50% reduction; Significance: P < 0.05

Parameter: ECAR (glycolysis, 20 h); Dnm2 HET: High; Dnm2 KO: ~50% of HET; Significance: P < 0.01

Parameter: OCR (oxidative phosphorylation, 20 h); Dnm2 HET: Moderate; Dnm2 KO: Lower; Significance: -

Parameter: OCR/ECAR ratio (20 h); Dnm2 HET: Low; Dnm2 KO: Higher; Significance: P < 0.01

Parameter: Glucose uptake (2-NBDG, 20 h); Dnm2 HET: High; Dnm2 KO: Suboptimal; Significance: P < 0.05

Parameter: Glut1 protein (20 h); Dnm2 HET: Up-regulated; Dnm2 KO: Lower; Significance: -

Parameter: Hk2 mRNA; Dnm2 HET: Induced; Dnm2 KO: Reduced; Significance: P < 0.05

Pathway: ERK phosphorylation; Status in Dnm2 KO: Enhanced

Pathway: JNK phosphorylation; Status in Dnm2 KO: Similar

Pathway: p38 phosphorylation; Status in Dnm2 KO: Similar

Pathway: Lck phosphorylation; Status in Dnm2 KO: Similar (normal)

Pathway: ZAP70 phosphorylation; Status in Dnm2 KO: Reduced

Pathway: Akt phosphorylation (T308, S473); Status in Dnm2 KO: Similar (intact)

Pathway: Tsc2 phosphorylation; Status in Dnm2 KO: Similar

Pathway: AMPKα phosphorylation; Status in Dnm2 KO: Decreased (not increased)

1. Dynamin 2 may have endocytosis-independent functions: Dynamin 2 also regulates actin polymerization at the immunological synapse (Gomez et al., 2005). While the authors focus on endocytosis, some effects could be due to non-endocytic roles of dynamin 2.

2. Pharmacological inhibitor alternatives: The authors used genetic knockout to avoid off-target effects of dynamin inhibitors (e.g., dynasore), but acknowledge that dynamin 2 deletion is a complete loss-of-function approach that may have developmental or compensatory effects.

3. Thymic egress confounding: Dnm2 KO mice have impaired thymic egress, which could affect peripheral T cell populations. The authors addressed this using adoptive transfer and FTY720 controls, but long-term effects of altered thymic selection cannot be completely excluded.

4. Causal relationship between c-Myc and mTORC1: The paper shows both pathways are affected, but the precise hierarchy (whether c-Myc loss causes mTORC1 reduction or vice versa) is not fully resolved. The authors note that Myc mRNA reduction occurs at 8 h when mTORC1 is still normal, suggesting c-Myc is upstream.

5. In vitro vs. in vivo differences: Some experiments (e.g., Seahorse metabolic analysis) were performed in vitro, which may not fully recapitulate the in vivo metabolic environment.

6. Cell type specificity: The study focused on CD4 and CD8 T cells; whether the same mechanism operates in other lymphocyte subsets (e.g., NKT cells, γδ T cells) is unknown.

7. Mechanism of c-Myc regulation: The paper does not fully elucidate how dynamin 2-dependent endocytosis regulates c-Myc expression—whether through specific endosomal signaling compartments, particular kinases, or post-transcriptional mechanisms.

8. No direct visualization of endosomal TCR signaling: While the study demonstrates functional requirement for dynamin 2, it does not directly image active signaling complexes in endosomes in primary T cells.

9. Autophagy as cause or consequence: Increased autophagy in Dnm2 KO cells could be a consequence of metabolic stress rather than a primary driver of the proliferation defect.

10. Human translation: All experiments were performed in murine T cells; the requirement for dynamin 2 in human T cells was not directly tested.

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