Modulation of Naive CD8 T Cell Response Features by Ligand Density, Affinity, and Continued Signaling via Internalized TCRs

Abstract

T cell response magnitudes increase with increasing antigenic dosage. However, it is unclear whether ligand density only modulates the proportions of responding ligand-specific T cells or also alters responses at the single cell level. Using brief (3 h) exposure of TCR-transgenic mouse CD8 T cells in vitro to varying densities of cognate peptide-MHC ligand followed by ligand-free culture in IL-2, we found that ligand density determined the frequencies of responding cells but not the expression levels of the early activation marker molecule, CD69. Cells with low glucose uptake capacity and low protein synthesis rates were less ligand-sensitive, implicating metabolic competence in the response heterogeneity of CD8 T cell populations. Although most responding cells proliferated, ligand density was associated with time of entry into proliferation and with the extent of cell surface TCR downmodulation. TCR internalization was associated, regardless of the ligand density, with rapidity of c-myc induction, loss of the cell cycle inhibitor p27kip1, metabolic reprogramming, and cell cycle entry. A low affinity peptide ligand behaved, regardless of ligand density, like a low density, high affinity ligand in all these parameters. Inhibition of signaling after ligand exposure selectively delayed proliferation in cells with internalized TCRs. Finally, internalized TCRs continued to signal and genetic modification of TCR internalization and trafficking altered the duration of signaling in a T cell hybridoma. Together, our findings indicate that heterogeneity among responding CD8 T cell populations in their ability to respond to TCR-mediated stimulation and internalize TCRs mediates detection of ligand density or affinity, contributing to graded response magnitudes.

Functionally significant metabolic differences between B and T lymphocyte lineages

“…25 Sorted B220+ and CD90+ cells were CFSE labelled and cultured on BMDC monolayers. 25 Sorted B220+ and CD90+ cells were CFSE labelled and cultured on BMDC monolayers.…”

“…Bone-marrow-derived dendritic cells (BMDCs) were cultured, as previously described. 25 Sorted B220+ and CD90+ cells were CFSE labelled and cultured on BMDC monolayers. Cells were rested in the incubator for 2 hr, and then images were taken on Nikon Zeiss microscope every 5 min for 2 hr.…”

T cell cytolytic capacity is independent of initial stimulation strength

“…In vitro studies have observed that reducing ligand affinity can decrease readouts of naive CD8 + T cell activation including calcium fluxes, kinase and transcription factor activity, surface receptor and cytokine expression, and proliferation5,11,13–23. Similarly, changing ligand concentration or costimulatory signals can affect T cell activation11,16,21,24–28. However, T cell activation assays differ between studies, with some reported as analog and others digital with respect to ligand affinity3,4,17,18,21,23,27,29,30.…”

Correlation of cell‐surface CD8 levels with function, phenotype and transcriptome of naive CD8 T cells

“…The mechanisms causing variability in homogeneous naive T‐cell populations are likely to be a mixture of the stochastic and the environmentally induced. We have previously reported that cell population variation in glucose metabolism and rate of protein synthesis could be involved in regulating the variation in the ability of CD8 T cells to respond to TCR‐mediated stimulation . Recent reports have traced the in vivo CD8 T‐cell response at single‐cell level, demonstrating that there is a strong heterogeneity in the numerical output of clonal expansion from individual T cells .…”

“…In the immune system, with respect to naive T cells, stochastic fluctuations in gene expressions of regulators of T‐cell receptor (TCR) signaling such as extracellular signal‐regulated kinase (ERK) kinases have been linked to the ability of T‐cell clones to sense differences in ligand affinities . We have recently shown that variation in baseline glucose uptake and rates of protein synthesis in naive T cells regulate their ability to respond to TCR‐mediated stimulation . Also, variation in expression of Src‐homology‐2 domain containing protein tyrosine phosphatases, which provides a negative feedback loop for TCR signaling, has been shown to regulate T‐cell sensitivity …”