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Related Experiment Video

Updated: Jan 13, 2026

Generation of Human Chimeric Antigen Receptor Regulatory T Cells
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CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28

Tetsushi Nishikawa1,2, Arata Takeuchi3, Hiroaki Machiyama1

  • 1Department of Immunology, Tokyo Medical University, Tokyo, Japan.

Communications Biology
|October 30, 2025
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Summary

Researchers visualized CD28-mediated signalosomes in chimeric antigen receptor (CAR)-T cells. This revealed how protein kinase C θ (PKCθ) accumulation enhances IL-2 production and tumor suppression, improving CAR-T cell therapy.

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Area of Science:

  • Immunology
  • Cell Biology
  • Biochemistry

Background:

  • Chimeric antigen receptor (CAR)-T cell therapy is advancing for various cancers.
  • Second-generation CARs use costimulatory domains like CD28, but endogenous T-cell receptors also provide parallel signals.
  • Understanding these distinct signaling pathways is crucial for CAR-T cell optimization.

Purpose of the Study:

  • To investigate and visualize CD28-mediated signalosomes in CAR-T cells.
  • To clarify the differences between direct CAR-induced and endogenous T-cell receptor-induced signaling.
  • To correlate signalosome dynamics with CAR-T cell efficacy.

Main Methods:

  • High-resolution imaging techniques were employed to visualize signalosomes.
  • CAR-T cells engineered with CD3ζ and CD28 (CD28ζ.CAR) were analyzed.
  • Accumulation of downstream kinase protein kinase C θ (PKCθ) at signalosomes was quantified.

Main Results:

  • CD28ζ.CAR-T cells showed intense and sustained accumulation of PKCθ at CAR signalosomes.
  • Endogenous CD28 on CAR-T cells extended the association with the CAR.
  • PKCθ assembly correlated significantly with IL-2 production and in vivo tumor suppression.

Conclusions:

  • CAR-T cell development requires analyzing intrinsic T-cell responses.
  • Signalosome dynamics, visualized through molecular imaging, are key indicators of CAR-T cell function.
  • Targeting signalosome assembly may enhance CAR-T cell antitumor activity.