Immunology

"Cholesterol homeostasis and lipid raft dynamics at the basis oftumor-induced immune dysfunction in chronic lymphocytic leukemia"

Chaja F. Jacobs, Fleur S. Peters, Elena Camerini, Gaspard Cretenet, Joanne Rietveld, Bauke V. Schomakers, Michel van Weeghel, Nico Hahn, Sanne G. S. Verberk, Jan Van den Bossche, Mirjam Langeveld, Fleur Kleijwegt, Eric Eldering, Noam Zelcer, Arnon P. Kater, and Helga Simon-Molas.

Introduction

..then we will talk about:

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1st Treatment Attempt

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Parameters

Autologous T-cell Based Therapies

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Results

Lipids - Metabolic Demand

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Discussion

Material And Methods

Connect cholesterol homeostasisAnd t cell function in cell

THREE CRUCIAL CULTURAL CONDITIONS

SAMPLES

ACTIVATION

• PBMCs (CLL Patients vs Age-Matched Healthy Donors)
• Anti-CD3 and CD28 antibodies (T cells receptors simulation)

Exogenous Cholesterol Importance

RATIONALE: Test the adaptability of CLL T cells from cholesterol stress

Pharmacological inhibitors to map cell's resilience

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confocal microscopy

Bodipy staining

to evaluate fatty acid oxidation

focusing on the metabolic route

external uptake and traffiking: U18666A internal synthesis: Simvastatin

FAO blue to measure FAO, crucial for mitochondrial health

to look at lipid storage organization

to observe lipid storage organization

CLL T cells have a greater vulnerability

RE S U L T S

The Core Problem: CLL T-Cells Fail to Import Cholesterol

Finding 1 (Fig. 1A): T-cells must import cholesterol to proliferate. Proliferation stops in cholesterol-free media (LPDS) for both HD and CLL T-cells. Finding 2 (Fig. 1E): CLL T-cells fail to build the import "gateway." Healthy T-cells (HD) upregulate the LDL Receptor (LDLR) upon activation. CLL T-cells fail to do this. Finding 3 (Fig. 1C/D): Accessible cholesterol is the limiting factor. Trapping cholesterol inside the cell (with U18666A) stops proliferation, mimicking starvation. This proves usable cholesterol is what controls cell division.

RESULTS

The Root Cause & The Consequence

The Cause (Fig. 2): RNA-sequencing (Fig. 2C, 2F) shows CLL T-cells fail to express "master switch" genes SREBF1 and SREBF2. These genes are required to build the LDLR "gateway" and control overall lipid metabolism.The Consequence (Fig. 3): The genetic failure is confirmed at the protein level (e.g., low FASN in Fig. 3B). This creates a fatal dependency: since CLL cells can't import cholesterol, they must make it. Proof (Fig. 3H): Blocking internal synthesis with NB598 stops CLL T-cell proliferation. Healthy T-cells are unaffected (they just import more).

RESULTS

The organization of lipids in the cytoplasm is different in T cells from CLL patients compared to HD

Fig. A: CLL T cells have significantly higher lipid accumulationFig. B: In CLL T cells, there’s more green Bodipy fluorescence, showing more lipid droplets in the cytoplasm compared to HD T cells. Fig. C: in CLL lipids are less associated with PLIN2

RESULTS

The lipidome of CLL T cells is characterized by low cholesterol and phospholipids, and an accumulation of TAG, compared to HD

Fig. A: no global differences at the baseline Fig. B: clear separation in lipid composition between CLL and HDFig. C: TAGs are increased after stimulation Fig. D: ATGL levels are much lower in CLL T cells, which means impaired lipolysis Fig. E: CLL T cells have lower FAO activity

DISCUSSION

T-cell dysfunction in CLL = Collapse of lipid metabolism

Two-problem model:Activation failure → Healthy T-cells use fatty acid oxidation (FAO) to activate. CLL T-cells have low FAO → can’t burn fat → weak activation. Proliferation failure → Requires cholesterol for building new cell membranes. CLL T-cells can’t import or synthesize cholesterol, and instead store fat as useless TAGs -> no cholesterol → no lipid rafts → no proliferation signal.

Root cause:Transcription factors regulating lipid metabolism (SREBP, PPAR, LXR) are turned off, creating a vicious cycle of lipid deficiency and T-cell exhaustion. CLL cells also steal lipids from the environment, worsening the defect.

Therapeutic Insight: Fixing the Metabolism

SOLUTION TO T-CELL FAILURE: RESTORE LIPID METABOLISM IN THE LAB

Strategies for CAR-T cell improvement:Re-fuel T-cells during culture: Add cholesterol and essential lipids → enhance proliferation and persistence. Re-program metabolism: Enhance FAO → stronger activation and memory. Genetically reactivate SREBP, PPAR, LXR → restore lipid processing.

Thank you

Adamo Gaia, Chirdo Greta, D'Ambrosio Paola, Ionta Elisa, Marrocco Martina, Notarangelo Letizia