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. 2000 Jan 17;191(2):335-46.
doi: 10.1084/jem.191.2.335.

Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers

M A Daniels  1 , S C Jameson
Affiliations

Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers

M A Daniels et al. J Exp Med. .

Abstract

Recent data using MHC/peptide tetramers and dimers suggests that the T cell coreceptors, CD4 and CD8, although important for T cell activation, do not play a direct role in facilitating T cell receptor (TCR) binding to multivalent MHC/peptide ligands. Instead, a current model proposes that coreceptors are recruited only after a stable TCR-MHC/peptide complex has already formed and signaled. In contrast, we show using multimeric class I MHC/peptide ligands that CD8 plays a critical (in some cases obligatory) role in antigen-specific TCR binding. T cell activation, measured by calcium mobilization, was induced by multimeric but not monomeric ligands and also showed CD8 dependency. Our analysis using anti-CD8 antibodies revealed that binding to different epitopes of CD8 can either block or augment TCR-MHC/peptide interaction. These effects on TCR binding to high-affinity agonist ligands were even more pronounced when binding to multimeric low-affinity ligands, including TCR antagonists, was studied. Our data have important implications for the role of CD8 in TCR binding to MHC/peptide ligands and in T cell activation. In addition, our results argue against the view that multimeric MHC/peptide ligands bind directly and solely to the TCR; rather, our data highlight a pivotal contribution of CD8 for this association.

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Figures

Figure 1
Figure 1
Binding of MHC/peptide multimers is dependent on the specificity of the TCR. (A) Lymph node cells from 2C and OT-I mice were depleted of B cells and CD4+ cells and stained for 1 h at 37°C with the indicated PE-conjugated multimers (Mult.). The mean fluorescence intensity (MFI) of 2C cells was 401 when stained with the SIY/Kb multimer versus 4 using the OVA/Kb multimer. MFI for OT-I cells was 5 for the SIY/Kb multimer and 326 with the OVA/ Kb multimer. (B) Staining as in A, except these cells were stained with the anti-CD8α antibody 53.6.7 in addition to the indicated multimer. Numbers represent the percentages of cells in respective quadrants.
Figure 2
Figure 2
Variable effect of CD8 antibodies on MHC class I multimer binding OT-I LN T cells. LN cells from an OT-I RAG-1−/− animal were stained with the indicated PE-conjugated multimer (Mult.) at 37°C for 2 h, with or without simultaneous staining with saturating amounts of the indicated FITC-conjugated anti-CD8 antibodies. (A) Cells were stained with OVA/Kb multimer (top) or SIY/Kb multimer (bottom), with no anti-CD8 antibody (open histogram), 53.6.7 (dotted histogram), or 3.168 (filled histogram). The effect of the antibodies CT-CD8a or 53.5.8 was similar to that of 3.168 for both multimers, and data using these antibodies is omitted for clarity. (B) The MFI of staining with the indicated multimer in the presence of titrated anti-CD8 antibodies.
Figure 2
Figure 2
Variable effect of CD8 antibodies on MHC class I multimer binding OT-I LN T cells. LN cells from an OT-I RAG-1−/− animal were stained with the indicated PE-conjugated multimer (Mult.) at 37°C for 2 h, with or without simultaneous staining with saturating amounts of the indicated FITC-conjugated anti-CD8 antibodies. (A) Cells were stained with OVA/Kb multimer (top) or SIY/Kb multimer (bottom), with no anti-CD8 antibody (open histogram), 53.6.7 (dotted histogram), or 3.168 (filled histogram). The effect of the antibodies CT-CD8a or 53.5.8 was similar to that of 3.168 for both multimers, and data using these antibodies is omitted for clarity. (B) The MFI of staining with the indicated multimer in the presence of titrated anti-CD8 antibodies.
Figure 4
Figure 4
CD8 effect on multimer dissociation. (A) OVA/Kb multimers were allowed to bind to OT-I RAG−/− cells for 2 h at 4°C in the presence or absence of the anti-CD8 antibodies, as indicated. (B) In the same experiment, OT-I cells that were stained with OVA/Kb multimers in the absence of anti-CD8 antibody as in A were washed twice and then incubated for an additional 2 h at 4°C with or without anti-CD8 antibodies as indicated.
Figure 3
Figure 3
Effects of temperature on multimer staining and effects of anti-CD8 antibodies. OT-I RAG-1−/− LN cells were stained at 4°C (1 h, left panels) or 37°C (1 h, right panels) with the SIY/Kb multimer (shaded) or OVA/Kb multimer (open) without anti-CD8 antibody (A) or with CD8 antibody 53.6.7 (B) or 3.168 (C). The block with 3.168 causes the OVA/Kb multimer to overlap the histogram for the negative control, SIY/Kb multimer.
Figure 6
Figure 6
Role of CD8 binding of multimeric altered peptide ligands. OT-I cells were incubated at 4°C for 2 h with Kb multimers containing OVA, SIY, G4, or E1 peptides in the absence of anti-CD8 antibodies (A) or in the presence of saturating amounts of 53.6.7 (B) or 3.168 (C). In the case of 3.168, the histograms with all the multimers overlap that of the negative control SIY/Kb multimer. Results are representative of four separate experiments.
Figure 5
Figure 5
Role of CD8 in multimer binding to 2C cells. 2C LN cells were depleted of B cells/CD4+ T cells and stained for 2 h at 4°C with PE-conjugated SIY/Kb multimer (A) or OVA/Kb multimer (B), with no anti-CD8 antibody (open histogram), 53.6.7 (dotted histogram), or 3.168 (filled histogram). The effect of the antibodies CT-CD8a or 53.5.8 was similar to that of 3.168 for both multimers, and data using these antibodies is omitted for clarity. In a separate experiment, SIY/Kb multimer staining of 2C T cells was determined in the absence (C) or presence of the anti CD8 antibodies 53.6 (D) or 3.168 (E). F, G, and H show CD8/FITC staining for the cells in C, D, and E, respectively. The multimer staining of gated CD8− (shaded) and CD8+ (open) populations revealed with 53.6.7 (I) or 3.168 (J) is also shown.
Figure 8
Figure 8
Ca2+ flux response in OT-I or 2C T cells induced by MHC/peptide monomers and multimers. (A) Ca2+ mobilization in OT-I RAG-1−/− LN cells treated with OVA/Kb multimers (blue line), free OVA peptide (red line), or OVA/Kb monomeric complexes (black line). In the case of the monomers, SA–PE was added at 8 min (arrow) and the sample reanalyzed for a further ∼8 min. The OVA/Kb multimer trace is taken from an 8-min time course and overlaid here as a representation of a responding population. (B and C) OT-I cells were pretreated with either 3.168 (B) or 53.6.7 (C) antibodies (black lines) before stimulation with OVA/Kb multimers or were exposed to SIY/Kb multimers (red lines) or OVA/Kb multimers (blue lines) in the absence of anti-CD8 antibodies. (D) In parallel, OT-I cells were stimulated by addition of anti-CD3 antibody 500.A2 (αCD3 antibody) plus cross-linking goat anti–mouse Ig, with (black line) or without (blue line) pretreatment with 3.168. 2C cells were stimulated with SIY/Kb or OVA/Kb multimers in the presence or absence of anti-CD8 antibodies 53.6.7 (E) or 3.168 (F). Response to the SIY/Kb multimer in the absence of any CD8 antibodies is shown in both panels (blue line). In E, the CD8+ (red line) and CD8− (black line) populations are delineated by 53.6.7. Exposure to OVA/Kb multimers in the presence of 53.6.7 (green line) served as a negative control in this response. In F, the CD8+ (black line) and CD8− (red line) populations are delineated by 3.168. All traces indicate the median of the responding population.
Figure 7
Figure 7
Density plot showing real time analysis of multimer binding and Ca2+ flux response. Indo-1AM–loaded OT-I RAG-1−/− LN cells were analyzed by FACS® for 1 min, at which time OVA/Kb (left panels) or SIY/Kb (right panels) PE-conjugated multimers (10 μg/ml) were added. Analysis of multimer binding (A) and Ca2+ mobilization (as reflected by changes in the fluorescence of the Indo-1 dye; (B) was determined for the same population of cells. Analysis of this and two other experiments indicates that 83–91% of OT-I cells mobilized Ca2+ in response to the OVA/Kb multimer.
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