Maturation-Dependent Licensing of Naive T Cells for Rapid TNF Production

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4+ and CD8+ single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs

Discrepancy between ELISPOT IFN-γ secretion and binding of A2/peptide multimers to TCR reveals interclonal dissociation of CTL effector function from TCR-peptide/MHC complexes half-life

Activation of CD8(+) cytolytic T lymphocytes (CTLs) by antigen is triggered by the interaction of clonotypic αβ T cell receptors (TCRs) with antigenic peptides bound to MHC class I molecules (pMHC complexes). Fluorescent multimeric pMHC complexes have been shown to specifically stain antigen-specific CTLs by directly binding the TCR. In tumor-infiltrating lymphocytes from a melanoma patient we found a high frequency of tyrosinase(368–376) peptide-specific cells as detected by IFN-γ ELISPOT, without detectable staining with the corresponding A2/peptide multimers. Surprisingly, these T cells were able to lyse tyrosinase(368–376) peptide-pulsed target cells as efficiently as other specific T cells that were stained by multimers. Analysis of the staining patterns under different conditions of incubation time and temperature revealed that these results were explained by major differences in TCR-multimeric ligand interaction kinetics among the clones. Whereas no direct quantitative correlation between antigenic peptide concentration required for CTL effector functions and equilibrium multimer binding was observed interclonally, the latter was profoundly affected by the kinetics of TCR-ligand interaction. More importantly, our data indicate that similar levels of T cell activation can be achieved by independent CD8(+) T cell clonotypes displaying different TCR/pMHC complex dissociation rates

In vitro membrane reconstitution of the T-cell receptor proximal signaling network

T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45, and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over a ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that coclustering TCR-Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs