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Genetic ablation approaches are routinely used to establish gene function, but these are not beyond limitations. The intracellularly expressed camelid single-domain antibodies (sdAbs), or nanobodies, can specifically disrupt molecular interactions and, therefore, help ascertain their role in the process of cellular differentiation. The approach has not been evaluated in differentiating T cells or the stimulating DCs during viral infection. We modified the intracellular response of Galectin-3 (Gal-3) using an α-Gal-3 intrabody (IB) in antigen-specific CD8 ⁺ T cells and assessed their differentiation patterns during a resolving Influenza A virus encoding SIINFEKL epitope (IAV: WSN-SIINFEKL). The α-Gal-3 IB expressors, compared to the cells genetically depleted of Gal-3, exhibit a significantly prolonged activation, enhanced proliferation, and elevated cytokine production following in vitro stimulation. The α-Gal-3 IB expressing cells show enhanced functionality and generate an efficient effector response, favoring the generation of memory precursor effector cells (MPECs: CD127 ^hi KLRG1 ^lo ) over short-lived effector cells (SLECs: CD127 ^lo KLRG1 ^hi ) in IAV-infected animals. Such a differentiation pattern results in improved differentiation of effector memory (CD44 ^hi CD62L ^lo CCR7 ^lo ) cells amongst the α-Gal-3 IB-expressing cells. The generated memory cells are efficiently recalled upon challenge infection with MHV68-SIINFEKL and help control the infection better. Therefore, modifying the intracellular response of Gal-3 using a sdAb could improve the primary and memory response of antigen-specific CD8 ⁺ T cells.

Author Summary

Genetic depletion of Gal-3 significantly increases the response of antigen-specific CD8 ⁺ T cells; whether this effect is attributable to exogenous or endogenous Gal-3 remains to be adequately elucidated. Using an intracellularly expressed α-Gal-3 sdAb that modulates the levels of bioavailable Gal-3, we study the differentiation pattern of CD8 ⁺ T cells during Influenza A virus infection. The α-Gal-3 intrabody (IB)-expressing CD8 ⁺ T cells, compared to their control counterparts, generate functionally superior effector and memory responses, and the recalled memory cells help control the infection better. The study establishes the utility of an α-Gal-3 IB as a potential strategy to improve antigen-specific primary and memory response of CD8 ⁺ T cells.