Aljohani, A. | bioRxiv Science

bioRxiv2026

ImmTACs overcome cytotoxic T cell suppression

Immune mobilizing monoclonal TCR against cancer (ImmTAC) are cancer therapeutics that activate T cells through recognition of a tumor-associated antigenic MHC/peptide complex. A first-in-class ImmTAC, Tebentafusp, is approved for the treatment of metastatic uveal melanoma. While clinical efficacy is thus established, the cellular mechanisms underpinning ImmTAC action are not fully resolved. Using a recently established experimental strategy to generate suppressed human primary cytotoxic T lymphocytes (CTL), we have investigated an ImmTAC that recognizes a peptide derived from the tumor associated antigen NY-ESO-1 in comparison to direct engagement of a TCR recognizing the same MHC/peptide complex. In response to endogenous antigen presentation, ImmTACs could elicit tumor cell cytolysis by suppressed CTL, but not IFN{gamma} secretion, in a manner dependent on the engager affinity for CD3{varepsilon}. ImmTACs enhanced the efficient execution of subcellular CTL polarization steps required for effective cytolysis and could trigger calcium signaling. These data establish that ImmTACs activate CTL similarly to direct engagement of a TCR by MHC/peptide and are likely to retain this capability under suppressive conditions such as in the tumor microenvironment.

immunology↗

bioRxiv2025

Interaction with tumor cell spheroids induces suppression in primary human cytotoxic T cells

Cytotoxic T lymphocytes (CTL) are key effectors in the anti-tumor immune response. However, their function is commonly suppressed in tumors in the form of exhausted CTL. Understanding molecular mechanisms of suppression and of therapeutics to overcome them is of substantial basic and translational importance yet hindered by limited access to large numbers of exhausted CTL in vitro. Here we use three-dimensional tissue culture to generate primary human CTL with suppressed function. Using a 21-antibody flow cytometry panel and determination of calcium signaling and cell couple maintenance, we show that these cells closely resemble exhausted CTL from tumors. For better understanding of in vitro human primary CTL as key tools in therapeutic development, before and after induction of suppression, we have determined the dependence of CTL function on technicalities of in vitro CTL generation, antigen dose and affinity across two T cell receptors and multiple tumor cell lines. We have investigated morphology and subcellular F-actin distributions of CTL as a key regulators of effector function. Primary human CTL formed cell couples with tumor target cells even in the absence of antigen. Yet, gradual stabilization of such cell couples was associated with increasing CTL effector function. Induction of suppression substantially destabilized CTL tumor cell couples. This comprehensive characterization of the phenotype of in vitro primary human CTL, including a suppressed state, should facilitate their wider use in basic and translational research.

immunology↗