Distinct influences of peptide-MHC quality and quantity on in vivo T-cell responses

Abstract

The strength of T-cell receptor (TCR) stimulation and subsequent T-cell response depend on a combination of peptide-major histocompatibility complex (pMHC) density and potency. By comparing two different pMHC at doses yielding similar proliferation in vivo, we have highlighted unexpected differences in the qualitative and quantitative effects of TCR ligand. Measurements of cytokine sensitivity and two-photon imaging of T cell-dendritic cell (T-DC) interactions reveal discrimination between comparably weak stimuli resulting from either decreased pMHC potency or pMHC density. In addition, TCR-induced genes in broad gene expression profiles segregate into two groups: one that responds to cumulative TCR signal and another that responds to pMHC quality, independent of quantity. These observations suggest that models of TCR ligand discrimination must account for disparate sensitivity of downstream responses to specific influences of pMHC potency.

Fig. 1.

Peptide-MHC density can compensate for potency in the induction of proliferation in vivo. Adoptively transferred 5C.C7 T cells were isolated from LNs 48 h after injection of (A) a 10-fold titration or (B and C) high (2 μg) or low (0.2 μg) doses of MCC or 102S peptide. (A) Percentage of cells that had divided was assessed using dilution of CFSE. (B) Representative flow plots and (C) pooled frequencies of ki67 expression. Shaded histograms represent the no-peptide control. Data are representative of at least three independent experiments.

Fig. 2.

Peptide-MHC potency dissociates IL-2 sensitivity from cumulative TCR signal. Adoptively transferred 5C.C7 T cells were isolated from LNs 48 h after injection of high (2 μg) or low (0.2 μg) doses of MCC or 102S peptide. 5C.C7 were stripped of cytokine and treated with the indicated concentrations of IL-2 for 10 min before fixation and staining for pSTAT5. Representative (A) flow plots with GMFI Inset and (B) dose–response curves. Data are representative of at least three independent experiments.

Fig. 3.

The affinity of TCR/pMHC interactions determines IL-2 and IL-2 receptor expression levels. (A) 5C.C7 division was assessed using dilution of CFSE 48 h after injection of doses ranging from 0.1 to 1 μg for MCC and 1 to 10 μg for 102S. The threshold ratio is the ratio of 1 μg for MCC and 10 μg for 102S. Samples from A were analyzed for (B and C) total CD25 and CD122 directly ex vivo or (D and E) IL-2 postrestimulation, compared with percentage of cells divided. Dashed lines (B–D) indicate the position of no-peptide controls. P values compare CD25, CD122, or IL-2 values between groups. Data are representative of at least three experiments.

Fig. 4.

The potency of pMHC is a greater influence on T-DC interactions than pMHC density after controlling for proliferation. 5C.C7 Ub-GFP T cells were imaged using two-photon microscopy on intact LN 15 hours after injection of the indicated peptide (High: 2μg, Low: 0.2μg). (A) Cumulative frequency plot for velocity is shown from one representative experiment of three. (B) Representative images are shown with circles indicating 5C.C7 interacting with CD11c-YFP DC (solid line indicates ≥300 sec). (C) Tabulated duration of interactions and (D) percent of time in contact with DC are shown. Contact duration was analyzed for two of three representative two-photon experiments. 5C.C7 surface expression of CD69 and CD62L (E) was assessed in LN cells isolated at the time of imaging. Shaded histograms represent the no-peptide control.

Fig. 5.

TCR-stimulated genes can be apportioned on the basis of sensitivity to distinct influences of pMHC potency. Adoptively transferred 5C.C7 T cells were isolated from LNs 48 h after injection of high (2 μg) or low (0.2 μg) doses of MCC or 102S peptide. Affymetrix microarray analysis of mRNA from sorted 5C.C7 T cells, with three independent samples per condition. TCR-regulated genes, with a difference in expression of twofold or more compared with low-dose 102S, were batched on the basis of similar expression due to (A) cumulative weak TCR stimulation or (B) pMHC potency.