LPS stimulates and Hsp70 down-regulates TLR4 to orchestrate differential cytokine response of culture-differentiated innate memory CD8+ T cells

• CD4+CD8+ thymocytes differentiate to innate memory CD8+ T cells in culture.
• The differentiated cells express TLR4 and show dual release of IL-10 and IFN-γ.
• Stimulation of TLR4 by LPS promotes IFN-γ release suppressing IL-10.
• Hsp70 induces TOLLIP down-regulating TLR4 and augmenting IL-10.

Nonconventional innate memory CD8+ T cells characteristically expressing CD44, CD122, eomesodermin (Eomes) and promyelocytic leukemia zinc finger (PLZF) were derived in culture from CD4+CD8+ double positive (DP) thymocytes of normal BALB/c and C57BL/6 mice. These culture-differentiated cells constitutively express toll-like receptor (TLR)4 and release interferon (IFN)-γ and interleukin (IL)-10. We show the TLR4-ligand lipopolysaccharide (LPS) stimulate the TLR and up-regulate IFN-γ skewing the cells towards type 1 polarization. In presence of LPS these cells also express suppressor of cytokine signaling (SOCS)1 and thus suppress IL-10 expression. In contrast, heat shock protein (Hsp)70 down-regulated TLR4 augmenting the anti-inflammatory cytokine IL-10. In association with IL-10 release IFN-γ was abrogated. The programmed cell death (PD)-1 mostly present in regulatory T cells was stimulated in these IL-10 producing cells by Hsp70 and not LPS indicating the cells can be driven to two contrast outcomes by the two TLR4 ligands. Our work provides a scope for in vitro monitoring of CD8+ T cells to decipher important immune therapeutic option during infection or sepsis.