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Comparative Study
. 2003 Nov 1;171(9):4493-503.
doi: 10.4049/jimmunol.171.9.4493.

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement

Samantha E Kerry  1 , Jennifer Buslepp, Lorraine A Cramer, Robert Maile, Lucinda L Hensley, Alma I Nielsen, Paula Kavathas, Barbara J Vilen, Edward J Collins, Jeffrey A Frelinger
Affiliations
Comparative Study

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement

Samantha E Kerry et al. J Immunol. .

Abstract

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.

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Figures

FIGURE 1
FIGURE 1
D227K mutation abrogates CD8 binding, but does not abrogate binding of MHC and TCR. A, Db/C9M and Db D227K/C9M tetramers were used to stain untransfected COS-7 cells or COS-7 cells transfected with CD8 α- and/or β-chains. Data are representative of two independent experiments. B, Monomers containing either gp33 or C9M peptides were measured for their ability to bind P14 TCR by SPR. Data are the average of two independent experiments. C and D, The ability of tetramers to bind live cells was measured by Scatchard analysis. The binding data were fitted to a standard Scatchard plot (C) and the dissociation constant (Kd) was calculated by the slope of the line (D). Kd values for Db/C9M and Db D227K/C9M differ significantly (p ≤ 0.05), as do Db/gp33 and Db D227K/gp33 (p ≤ 0.005).
FIGURE 2
FIGURE 2
CD8 engagement increases CD8+ T cell proliferation, IFN-γ and TNF-α production, and CTL activity. A, CD8+ P14 splenocytes were stimulated with Db/gp33 (●) and Db D227K/gp33 (○) or control (x) tetramers, then measured for their ability to proliferate in vitro. Each concentration was assayed in triplicate. All error bars are ±SEM. Data are representative of five independent experiments. B, In vivo stimulation of splenocytes and proliferation based upon CFSE staining. The percentage of divided cells was calculated using Modfit software. Proliferation by Db D227K/gp33-stimulated CD8+ T cells is significantly decreased in vivo (*, p ≤ 0.05) compared with cells stimulated with Db/gp33 tetramers. Each group contained four mice and the data are representative of two independent experiments. C, Production of IFN-γ and TNF-α by in vitro-stimulated CD8+ T cells. IFN-γ and TNF-α production were significantly decreased (***, p ≤ 0.001 and **, p ≤ 0.01) without CD8 engagement compared with stimulation with wild-type tetramer. Each sample was assayed in triplicate by a cytometric bead array and normalized to the amount of cytokine induced by Db/gp33. Data are representative of two independent experiments. D, CTL activity of tetramer-stimulated P14 cells. Target cells were pulsed with relevant (●, ○) and irrelevant peptide (▲,△). Db D227K/gp33-stimulated cells are shown with open symbols, while Db/gp33-stimulated cells are shown with filled symbols. Triplicate wells at each E:T ratio were averaged. Data are representative of two independent experiments.
FIGURE 3
FIGURE 3
Higher affinity MHC-TCR interaction overcomes need for CD8 engagement. A, CD8+ P14 splenocytes were stimulated with Db/C9M (■), Db D227K/C9M (□), or control (×) tetramers, then measured for their ability to proliferate in vitro. Samples were assayed in triplicate, and the average was plotted with the error bars indicating ±SEM. Data are representative of five independent experiments. B, Measurement of in vivo proliferative ability of cells stimulated with Db/C9M or Db D227K/C9M. Db D227K/C9M-stimulated CD8+ T cells illustrate similar proliferative ability, as compared with cells stimulated with Db/C9M tetramers (*, p ≤ 0.05). In vivo proliferation was performed using four mice per group, the percentage of divided cells was calculated as in Fig. 2, and the data are representative of two independent experiments. C, IFN-γ production by Db D227K/C9M-stimulated cells is significantly lower as compared with Db/C9M stimulation (***, p ≤ 0.001). Each sample was assayed in triplicate and the amount of cytokines produced was normalized to cytokine levels produced by Db/gp33 stimulation. Data are representative of two independent experiments. D, CTL activity of tetramer-stimulated P14 cells. Target cells were pulsed with relevant (■,□) and irrelevant peptide (▲,△). Db D227K/C9M-stimulated cells are shown with open symbols, while Db/C9M-stimulated cells are shown with filled symbols. The average of three replicates per ratio is plotted, and data are representative of three independent experiments.
FIGURE 4
FIGURE 4
CD8 engagement with MHC/ TCR induces altered signaling patterns, as compared with MHC/TCR alone. A, CD8 P14 splenocytes were stimulated for 3 min with tetramers at 37°C, and phosphotyrosine activity was detected by Western blot. Cells were unstimulated (US) or stimulated with Db/gp33 (lane 1), Db D227K/gp33 (lane 2), Db/C9M (lane 3), or Db D227K/C9M (lane 4). Data are representative of three independent experiments. B, Quantitation of band intensity of tetramer-stimulated cells. To measure the intensity of the bands, pixel volume of identical areas of the gel were determined for each lane and corrected for local area background. Two bands missing in the Db D227K stimulated and one control band are plotted. C, Calcium mobilization of cells treated with indicated tetramers. Data are representative of two independent experiments.
FIGURE 5
FIGURE 5
CD8 is present in mi-crodomains without engagement by MHC. A, CD8+ P14 splenocytes were stained with anti-CD8α and Db/C9M (top), Db D227K/C9M (middle), or Db tetramer containing an irrelevant peptide (Db/flu, bottom). Tetramer staining is shown in red, CD8 staining in green, and colocalization of tetramer and CD8 in yellow. CD8 colocalizes with tetramer in discrete patches (foci, arrows) with and without tetramer engagement of CD8. Data are representative of at least three independent experiments. B, Calculated colocalization of CD8 and tetramer.
FIGURE 6
FIGURE 6
CD8 is in close proximity to TCR on the cell surface. P14 splenocytes were labeled with the indicated Abs and analyzed by FACS for FRET signal. A, The addition TCR-TRITC to CD8-FITC stained cells produced significantly increased FRET, thus indicating that CD8 and TCR are closely associated (p ≤ 0.01). B, Addition of Db/C9M (blue), but not Db D227K/C9M (red) or irrelevant tetramer (black) significantly increases FRET of cells prelabeled with both TCR-TRITC and CD8-FITC (p ≤ 0.001). Data are representative of three independent experiments.
FIGURE 7
FIGURE 7
Higher affinity MHC-TCR interactions produce an additive activation signal over time. A, Purified P14 CD8+ cells were stimulated with Db/gp33 or Db D227K/gp33 tetramers and activation of p56Lck kinase was measured. Db D227K/gp33 stimulated significantly higher kinase activity than irrelevant or no tetramer (**, p < 0.005). However, Db/gp33 induces significantly greater p56Lck activity than Db D227K/gp33 (*, p < 0.005). Data are representative of three independent experiments. B, Purified P14 CD8+ cells were stimulated with Db/C9M or Db D227K/C9M tetramers for 15 min or 2 h and washed away. Control cells received tetramer stimulation for the entire course of the assay (36 h). Data are representative of two independent experiments.
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