TCR Signaling for Initiation and Completion of Thymocyte Positive Selection Has Distinct Requirements for Ligand Quality and Presenting Cell Type

Abstract

Thymocyte selection involves signaling by TCR engaging diverse self-peptide:MHC molecule ligands on various cell types in the cortex and medulla. Here we separately analyze early and late stages of selection to better understand how presenting cell type, ligand quality, and the timing of TCR signaling contribute to intrathymic differentiation. TCR transgenic CD4+CD8+ thymocytes (double positive (DP)) from MHC-deficient mice were stimulated using various presenting cells and ligands. The resulting CD69high cells were isolated and evaluated for maturation in reaggregate cultures with wild-type or MHC molecule-deficient thymic stroma with or without added hemopoietic dendritic cells (DC). Production of CD4+ T cells required TCR signaling in the reaggregates, indicating that transient recognition of self-ligands by DP is inadequate for full differentiation. DC bearing a potent agonist ligand could initiate positive selection, producing activated thymocytes that matured into agonist-responsive T cells in reaggregates lacking the same ligand. DC could also support the TCR signaling necessary for late maturation. These results argue that despite the negative role assigned to DC in past studies, neither the peptide:MHC molecule complexes present on DC nor any other signals provided by these cells stimulate only thymocyte death. These findings also indicate that unique epithelial ligands are not necessary for positive selection. They provide additional insight into the role of ligand quality in selection events and support the concept that following initiation of maturation from the DP state, persistent TCR signaling is characteristic of and perhaps required by T cells.

Delineation of Signals Required for Thymocyte Positive Selection

“…This finding suggests that a proportion of cells undergoing coreceptor dulling induced by negatively selecting ligands in vitro may have paradoxically initiated differentiation into one of the mature thymocyte subpopulations. This conclusion is consistent with the results showing enhanced positive selection of thymocytes stimulated in vitro with negatively selecting ligands and then assembled in MHC-disparate thymus reaggregation cultures (12).…”

Maturational stage-dependent thymocyte responses to TCR engagement

“…Deletion of TCR-Vb6 + cells was not detected before the intermediate subset, and was complete by SP [44]. Negative selection following initiation of selection was later supported in reaggregate cultures, in which positive selection of MHC class II-restricted transgenic thymocytes could be initiated by a strong agonist peptide presented by dendritic cells [30]. These thymocytes could then complete positive selection if the agonist was removed after the intermediate stage of selection, or would undergo deletion with continued strong agonist interaction.…”

“…Alternatively, it had been suggested that results of TCR-mediated recognition events, positive or negative selection, are equally dependent on the maturation state of the thymocytes [27][28][29]. Further studies showed that intermediate thymocytes induced in vitro [21], or isolated in vivo [22] could be driven to maturity [22,30] or be negatively selected only after the intermediate stage [28,30]. Thus, these studies suggested that thymocyte responses to TCR-mediated recognition events are maturation stage dependent.…”

Perspectives for Computer Modeling in the Study of T Cell Activation

“…T cells endowed mainly with receptors of intermediate affinity for self pMHC ligands pass the filters of positive/negative selection (Alam et al 1996;Savage et al 1999;Savage and Davis 2001). Beyond the role of the intrinsic affinity of the TCR for selecting ligands in the maturation process, it is also clear that T cells modify the intracellular components of the signaling apparatus to tune the amplitude of their signaling response induced by self-ligand during thymocyte development, extinguishing responses to weak ligands while not constraining responses to strong ligands (Hogquist et al 1994;Lucas et al 1999;Yasutomo et al 2000;Hogquist 2001;Starr et al 2003).…”