T cell activation enhancement by endogenous pMHC acts for both weak and strong agonists but varies with differentiation state

Abstract

T cells are extremely sensitive in their ability to find minute amounts of antigenic peptide in the midst of many endogenous peptides presented on an antigen-presenting cell. The role of endogenous peptides in the recognition of foreign peptide and hence in T cell activation has remained controversial for CD8(+) T cell activation. We showed previously that in a CD8(+) T cell hybridoma, nonstimulatory endogenous peptides enhance T cell sensitivity to antigen by increasing the coreceptor function of CD8. However, others were not able to detect such enhancement in naive and activated CD8(+) T cells. Here, we show that endogenous peptides substantially enhance the ability of T cells to detect antigen, an effect measurable by up-regulation of activation or maturation markers and by increased effector function. This enhancement is most pronounced in thymocytes, moderate in naive T cells, and mild in effector T cells. The importance of endogenous peptides is inversely proportional to the agonist activity of the stimulatory peptide presented. Unlike for CD4(+) T cells, the T cell receptor of CD8(+) T cells does not distinguish between endogenous peptides for their ability to enhance antigen recognition.

Figure 1.

Nonstimulatory peptides increase antigen recognition by T cells at different differentiation states. RMA-S cells were loaded with various amounts of OVA alone or OVA plus nonstimulatory peptide. The RMA-S cells were allowed to interact with thymocytes (A, D, and G), naive T cells (B, E, and H), or effector CTLs (C, F, and I) for 5 h. The cells were stained with anti-CD8 and anti-CD69, and the level of CD69 was assessed by flow cytometry. The results are shown as percentage of CD69⁺ cells (A, B, and C) or as mean fluorescent intensity (MFI) of CD69 staining (D, E, and F) from CD8 gated cells at different OVA-K^b concentrations. For thymocytes, the overall population behaves uniformly, and the graphs for MFI and percent CD69^hi populations look similar. For naive cells, CD69 up-regulation is bimodal. However, in the presence of nonstimulatory peptide, the MFI of the CD69^hi cells is higher than in the absence of nonstimulatory peptides, and therefore the two groups display bigger differences when presented as MFI as opposed to percent CD69^hi. Example histograms (G, H, and I) are shown for each of the cell subsets. In the histograms, the dashed lines correspond to thymocytes or T cells incubated with RMA-S cells without added peptide, the dotted lines to cells incubated with RMA-S cells loaded with OVA alone, and the solid black lines to cells incubated with RMA-S cells loaded with OVA together with a large excess of the nonstimulatory VSV peptide. The example histograms have been chosen in such a way that the OVA-alone group displays more antigen than the group with the nonstimulatory peptide. (Fig. S1 shows an OVA-K^b–matched comparison.) The results are representative of at least three independent experiments.

Figure 2.

The effect of nonstimulatory peptides is most pronounced at early time points. RMA-S cells were loaded with various amounts of OVA alone or OVA plus VSV. The RMA-S cells were allowed to interact with naive T cells for 2 (A), 7 (B), or 20 h (C). The cells were stained with anti-CD8 and anti-CD69, and the level of CD69 was assessed by flow cytometry. The results are shown as percentage of CD69⁺ cells from CD8 gated cells at different OVA-K^b concentrations. The results are representative of four independent experiments.

Figure 3.

The effect of nonstimulatory peptides is increased with weaker agonists. RMA-S cells were loaded with various amounts of OVA, Q4 or T4 alone, or the same peptides plus VSV, respectively. The RMA-S cells were allowed to interact with naive T cells for 24 (A) or 5 h (B), or with effector cells for 3 h (C). The cells were stained with anti-CD8 in combination with anti-CD25 (A), anti-CD69 (B), or anti-Vα2 (C), and the level of these molecules was assessed by flow cytometry. The results are shown as percentage of CD25⁺ (A) and percentage of CD69⁺ (B) CD8 cells at different OVA, Q4, or T4-K^b concentrations. The TCR down-regulation data (C) are shown as percentages of Vα2 expression on the surface of CD8 cells incubated with RMA-S cells presenting different amounts of OVA, Q4, or T4-K^b compared with cells incubated with RMA-S cells in the absence of an exogenously added peptide. The results are representative of four independent experiments.

Figure 4.

The ability of nonstimulatory peptides to enhance antigen recognition is independent of the agonist strength. RMA-S cells were loaded with various amounts of OVA (A), Q4 (B), or T4 (C) alone or together with a nonstimulatory peptide. The RMA-S cells were allowed to interact with DP thymocytes for 5 h. The cells were stained with anti-CD8 and anti-CD69, and the level of CD69 was assessed by flow cytometry. The results are shown as percentage of CD69⁺ CD8 gated cells at different OVA, Q4, or T4-K^b concentrations. The results are representative of at least two independent experiments.

Figure 5.

The effect of nonstimulatory peptides on antigen recognition depends on quantity but not quality of the nonstimulatory pMHC. RMA-S cells were loaded with OVA peptide, washed, and aliquoted to different groups. Nonstimulatory peptides were titrated on the antigen-loaded cells and, after incubation, washed away. The cells were allowed to interact with DP thymocytes for 2 h. The cells were stained with anti-CD8 and anti-CD69, and the level of CD69 was assessed by flow cytometry. The results are shown as percentage of CD69⁺ CD8 gated cells in the presence of different numbers of K^b molecules. The effect of the nonstimulatory peptides without any OVA is shown surrounded by a box, but using the same symbols as for those with added OVA. The results are representative of at least three independent experiments. Fig. S1 shows histograms of CD69 up-regulation by RMA-S cells matched for low expression of OVA-K^b in the presence or absence of excess nonstimulatory peptide-K^b.

Figure 6.

Nonstimulatory peptides enhance cytokine production. RMA-S cells were loaded with various amounts of OVA alone or together with VSV. The RMA-S cells were allowed to interact with naive T cells (A) or effector cells (B) for 5 h (A and B). The cells were first stained with anti-CD8, and then intracellularly with anti–IL-2 (A) or anti–IFN-γ (B), and the cytokine level was assessed by flow cytometry. The results are shown as percentage of cytokine⁺ cells at different OVA-K^b concentrations. The results are representative of eight independent experiments.

Figure 7.

Nonstimulatory peptides enhance CTL killing of target cells. RMA-S cells were loaded with various amounts of OVA or T4 alone or together with VSV. The Cy5-labeled RMA-S cells were allowed to interact with effector cells for 17 h. Cell death was determined by death-associated changes in the forward- and side-scatter properties among the RMA-S cell population (Cy5⁺). Results are shown as percentage of live RMA-S cells for different OVA or T4-K^b concentrations and are representative of four independent experiments.

Figure 8.

Nonstimulatory peptides affect duration of T–APC conjugates. RMA-S cells were labeled with Cy5 and loaded with various amounts of OVA or T4 alone or together with VSV. The RMA-S cells were allowed to interact with naive T cells for the indicated times. The cells were fixed and stained for CD8, and cell conjugate formation was assessed by flow cytometry based on simultaneous staining for CD8 and Cy5. Results are shown as percentage of cell conjugates among CD8 cells for different OVA or T4-K^b concentrations at 20- (A) and 60-min (B) time points. The data are representative of at least four independent experiments.