Opto-CD28-REACT: Optogenetic co-stimulatory receptor activation on non-engineered human T cells

Anna K Ehret^1,2,3,4,5Sara Hartmann^1,2,3,4,5Pavel Salavei^1,2,6Virginia Andreani^1,4Nicole Gensch^1,2,6Laura Gámez-Díaz^1,4Wolfgang W. Schamel^1,2,3,4,7*

• ¹CIBSS Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany
• ²BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany
• ³Faculty of Biology, University of Freiburg, Freiburg, Germany
• ⁴Centre for Chronic Immunodeficiency (CCI), Medical Centre Freiburg and Faculty of Medicine, University of Freiburg, Freiburg, Germany
• ⁵Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany
• ⁶Core Facility Signalling Factory & Robotics, Centre for Biological Signaling Studies (BIOSS), University of Freiburg, Freiburg, Germany
• ⁷Centre for Cell and Gene Therapy Freiburg (CGF), Medical Centre Freiburg and Faculty of Medicine, University of Freiburg, Freiburg, Germany

T cell activation is a highly regulated process requiring both antigen recognition via the T cell receptor (TCR) and co-stimulatory signaling, notably through the co-stimulatory receptor CD28. Here, we introduce an optogenetic platform for reversible and tunable full activation of human T cells that does not require genetic modification. We engineered opto-CD28-REACT, a recombinant protein comprising an anti-CD28 single-chain variable fragment, GFP, and the PhyB-interacting factor PIF6. This construct binds CD28 and thereby attaches PIF6 to CD28. U and enables light-controlled receptor clustering via interaction with PhyB tetramers. Upon red light (630 nm) illumination, opto-CD28-REACTPIF6 engages withbinds to PhyB tetramer-coated beads,, triggering CD28 signaling that can be rapidly attenuated by far-red light (780 nm) in two minutes. We show that opto-CD28-REACT synergizes with opto-CD3ε-REACT -a complementary optogenetic tool targeting the TCR complexto induce light-dependent activation of both Jurkat cells and primary human T cells. Co-stimulation through both opto-REACT systems promotes robust ERK phosphorylation, upregulation of the activation markers (CD69 and, CD25), IL-2 secretion, and T cell proliferation, reaching levels comparable similar to conventional antibody-mediated stimulation. This strategy enables precise optical control over TCR and CD28 signaling in non-genetically modified T cells, offering a powerful approach for dissecting the regulatory dynamics of T cell activation and advancing applications in synthetic immunology and immunotherapy.