Nanomatrix-based TCR stimulation supports CD4+ but not CD8+ T cell effector functions

Introduction In vitro T cell stimulation is commonly used to model physiological TCR-pMHC engagement to study T cell activation, signaling, proliferation, and differentiation. Classical stimulation methods use anti-CD3 antibodies combined with anti-CD28 antibodies, in soluble or plate-bound forms. This promotes TCR crosslinking and robust activation. Here, we evaluated TransAct, a soluble nanomatrix-conjugated antibody reagent, as an alternative to conventional antibody-based stimulation methods. Methods Both CD4 + and CD8 + T cells were stimulated with anti-CD3/CD28 antibodies or TransAct, and proliferation, cytokine production, and the expression of activation markers were assessed by flow cytometry. Additionally, TCR polarization was analyzed by ImageStream analysis. Results Whereas T cell proliferation was comparable for CD4 + T cells stimulated with TransAct or anti-CD3/CD28 antibodies, it was dramatically reduced in TransAct-stimulated CD8 + T cells compared to the antibody treatment. Stimulation with antibodies elicited high levels of IL-2, IFN, and TNF in both subsets, whereas TransAct generally triggered lower cytokine production, with particularly attenuated responses in CD8 + T cells. The early activation markers CD69 and CD25 were upregulated by TransAct in CD4 + T cells and, to a lesser extent, in CD8 + T cells. However, the late activation marker HLA-DR, activation-associated inhibitory receptors (PD-1, CTLA-4), and FoxP3 were more strongly induced by antibodies, particularly in CD8 + T cells. Imaging flow cytometry analysis confirmed altered spatial organization of the TCR complex after TransAct stimulation compared to antibody stimulation. Discussion These findings indicate that TransAct provides effective, relatively gentle activation of CD4 + T cells but delivers suboptimal activation signals to CD8 + T cells compared to classical plate-bound antibody stimulation.