Soluble peptide-MHC monomers cause activation of CD8+ T cells through transfer of the peptide to T cell MHC molecules

Abstract

T cell receptor (TCR)-mediated activation of CD4(+) T cells is known to require multivalent engagement of the TCR by, for example, oligomeric peptide-MHC complexes. In contrast, for CD8(+) T cells, there is evidence for TCR-mediated activation by univalent engagement of the TCR. We have here compared oligomeric and monomeric L(d) and K(b) peptide-MHC complexes and free peptide as stimulators of CD8(+) T cells expressing the 2C TCR. We found that the monomers are indeed effective in activating naive and effector CD8(+) T cells, but through an unexpected mechanism that involves transfer of peptide from soluble monomers to T cell endogenous MHC (K(b)) molecules. The result is that T cells, acting as antigen-presenting cells, are able to activate other naive T cells.

Figure 1

Activation of CD8⁺ T cell clones by class I MHC monomers and oligomers. (A–C) Response of a T cell clone expressing the 2C TCR (L3.100) to MHC monomers, oligomers, and free peptide. (A) TCR down-regulation in response to 3-h incubation with SIY-K^b oligomer (filled squares), SIY-K^b monomer (filled circles), and SIY peptide alone (open circles). Mean fluorescence intensity (MFI) shown on y axis. (B) CD69 up-regulation (MFI) and (C) CD25 up-regulation (MFI) in response to the same treatment as A. (D and E) Specificity of the response. (D) TCR down-regulation (MFI) is observed for L3.100 incubated for 3 h with SIY-K^b monomers (circles) but not for nonspecific OVA-K^b (inverted triangles). (E) Response of a T cell clone specific for OVA-K^b complex (4G3) shows TCR down-regulation (MFI) after 3 h of incubation with OVA-K^b monomers (inverted triangles), but not with SIY-K^b (circles).

Figure 2

Activation of naïve CD8⁺ T cells by class I MHC monomers and oligomers. (A and B) Response of naïve T cells expressing the 2C TCR to MHC monomers, oligomers, and free peptide. (A) TCR down-regulation in response to 2-h treatment with SIY-K^b oligomer (filled squares), SIY-K^b monomer (filled circles), and SIY peptide alone (open circles). Mean fluorescence intensity (MFI) shown on y axis. (B) CD69 up-regulation (MFI) in response to the same treatment. (C) Proliferation of naïve 2C T cells measured by ³H-thymidine incorporation 3 days after treatment in response to SIY-K^b monomer (filled circles), SIY peptide alone (open circles), denatured (boiled) SIY-K^b monomer (X), and nonspecific OVA-K^b monomer (inverted triangles). (D) TCR down-regulation (MFI) of CD8⁺ (circles) or CD8⁻ (diamonds) populations present in purified naïve 2C T cells in response to SIY-K^b monomers (filled symbols) or SIY peptide alone (open symbols). (E) Maturation of naïve 2C T-cells in response to MHC monomers, oligomers, and free peptide. Naïve 2C T cells were stimulated with immobilized anti-CD3 antibody (10 μg/ml), SIY-K^b oligomers, SIY-K^b monomers, or free SIY peptide for 3 days. Intracellular IFN-γ production was measured after 6-h restimulation by SIY peptide (bold line). The shaded area corresponds to isotype control staining.

Figure 3

Two potential mechanisms for activation of T cells by MHC monomers. (A) Binding of monomeric peptide–MHC complexes to cell-surface T cell receptors directly leads to activation (direct engagement model). (B) Transfer of peptide from the soluble MHC molecules to endogenous MHC molecules expressed by the T cell indirectly leads to activation of another T cell through interaction with peptide–MHC complexes on the surface of the first cell (representation model). Binding of soluble peptide–MHC to cell-surface TCR (or CD8) could facilitate peptide transfer, although other mechanisms are possible.

Figure 4

CD8⁺ T cell activation by MHC monomers requires endogenous MHC proteins. 2C T cells expressing K^b, but not L^d, are activated by soluble monomers of the syngeneic SIY-K^b but by not the potent alloantigen QL9-L^d. (A) Response of naïve 2C T cells to 3 h of incubation with peptide-pulsed APCs. T2 cells expressing K^b were pulsed with OVA (inverted triangles) and SIY (circles) peptides, and T2 cells expressing L^d were incubated with QL9 peptide (diamonds). TCR mean fluorescence intensity (MFI) shown on y axis. (B) Binding of fluorescein-labeled SIY-K^b (circles), OVA-K^b (inverted triangles), or QL9-L^d (diamonds) to naïve 2C T cells. Cell-associated fluorescence was measured after incubation with MHC monomers for 30 min at 4°C, washing, and fixation with paraformaldehyde. (C and D) Response of naïve 2C T cells to 3 h incubation with SIY-K^b (circles), OVA-K^b (inverted triangles), or QL9-L^d (diamonds) monomers. (C) TCR down-regulation (percentage of maximum TCR expression), and (D) CD69 up-regulation (MFI). (E) TCR down-regulation of naïve 2C T cells in response to 6-h incubation with plate-bound and soluble monomers or free peptide. Open bars represent SIY or SIY-K^b; shaded bars represent QL9 or QL9-L^d.

Figure 5

Lack of activation of H2-K^b−/−D^b−/− T cells by soluble MHC monomers. T cells expressing the 2C TCR, but not H2-K^b/D^b, are unable to be efficiently activated by soluble MHC monomers. (A) Binding of fluorescein-labeled MHC monomers to naïve 2C T cells (filled symbols) and 2C⁺, K^b−/−D^b−/− T cells (open symbols). Binding is shown for SIY-K^b (circles) and OVA-K^b (inverted triangles). MFI shown on y axis. (B) Bar graph showing the percent of TCR down-regulation for treatment of naïve 2C T cells (shaded bars) and 2C⁺, K^b−/−D^b−/− T cells (open bars) with peptide-pulsed T2-K^b cells and soluble SIY-K^b monomer. (C) Concentration dependence of TCR down-regulation (MFI) response in K^b+/+D^b+/+ (filled symbols) and K^b−/−D^b−/− (open symbols) naïve 2C T cells after 3-h incubation with soluble SIY-K^b (circles) and OVA-K^b (inverted triangles).

Figure 6

T cell activation by uptake and representation of antigen. Naïve GFP⁺ 2C T cells were activated by exposure to GFP⁻ 2C T cells that previously had been treated with MHC monomers. (A and B) GFP⁻ cells were treated with control media or SIY-K^b for 3 h, washed, and then GFP⁺ cells were added to the GFP⁻ cells in fresh media. The cells were incubated together for an additional 3 h at 37°C. (A) TCR down-regulation and (B) CD69 up-regulation. The horizontal scale is GFP fluorescence level which distinguishes the directly treated (GFP⁻) and indirectly exposed (GFP⁺) T cells. (C and D) Response of directly treated GFP⁺ 2C T cells. (C) TCR down-regulation by GFP⁺ 2C T cells in response to 3-h incubation with SIY-K^b (filled circles) or SIY peptide (open circles). MFI shown on y axis. (D) CD69 up-regulation (MFI) by GFP⁺ 2C T cells in response to the same treatment. (E and F) Response of indirectly exposed GFP⁺ 2C T cells. (E) TCR down-regulation (MFI) by GFP⁺ 2C T cells incubated for 3 h with GFP⁻ 2C T cells that had been pretreated with SIY-K^b (filled circles) or SIY peptide (open circles). (F) CD69 up-regulation (MFI) of GFP⁺ 2C T cells in response to the same stimuli.