Interaction between the CD8 coreceptor and major histocompatibility complex class I stabilizes T cell receptor-antigen complexes at the cell surface

Abstract

The off-rate (k(off)) of the T cell receptor (TCR)/peptide-major histocompatibility complex class I (pMHCI) interaction, and hence its half-life, is the principal kinetic feature that determines the biological outcome of TCR ligation. However, it is unclear whether the CD8 coreceptor, which binds pMHCI at a distinct site, influences this parameter. Although biophysical studies with soluble proteins show that TCR and CD8 do not bind cooperatively to pMHCI, accumulating evidence suggests that TCR associates with CD8 on the T cell surface. Here, we titrated and quantified the contribution of CD8 to TCR/pMHCI dissociation in membrane-constrained interactions using a panel of engineered pMHCI mutants that retain faithful TCR interactions but exhibit a spectrum of affinities for CD8 of >1,000-fold. Data modeling generates a "stabilization factor" that preferentially increases the predicted TCR triggering rate for low affinity pMHCI ligands, thereby suggesting an important role for CD8 in the phenomenon of T cell cross-reactivity.

Fig. 1. Mutation of the HLA A2 α2 or α3 domain can abrogate, decrease, or increase the interaction with CD8 without affecting the interaction between the α1/α2 peptide-binding domain and the TCR

Biotinylated pMHCI monomers were immobilized onto a streptavidin-coated BIAcore chip as described under “Experimental Procedures.” Serial dilutions of either sCD8αα wild type or soluble A6 Tax TCR in HBS-EP buffer were flowed over the chip to generate kinetic data. Data were analyzed using BIAeval, Excell, and Origin version 6.1 (Microcal software). K_D values were calculated both by linear Scatchard plots and non-linear analysis assuming 1:1 Langmuir binding (A + B⇆AB) using non-linear curve fitting to the equation AB = B × AB_max/(K_D + B). A, equilibrium binding of sCD8αα interacting with either D227K/T228A HLA A2 (227/8KA), A245V HLA A2, wild type HLA A2 (wildtype), Q115E HLA A2, or A2/K^b α3 domain fusion (A2/Kb) folded around the HTLV-1 Tax epitope. B, summary of sCD8αα/pMHCI affinity measurements obtained for pMHCI monomers bearing different mutations and folded around three different epitopes. DT227/8KA, D227K/T228A. C, the K_D calculated for the interaction between the A6 Tax TCR and either D227K/T228A HLA A2 (DT227/8KA), A245V HLA A2, wild type HLA A2, Q115E HLA A2, or A2/K^b α3 domain fusion folded around the HTLV-1 Tax epitope was shown to be 2.6 ± 0.4, 2.5 ± 0.3, 2.5 ± 0.3, 2.7 ± 0.4, and 2.5 ± 0.2 μm respectively. In all cases indicated errors are S.D. from the mean of three separate experiments. Data shown are from just one representative experiment (hence the values differ slightly from the averages given above).

Fig. 1. Mutation of the HLA A2 α2 or α3 domain can abrogate, decrease, or increase the interaction with CD8 without affecting the interaction between the α1/α2 peptide-binding domain and the TCR

Biotinylated pMHCI monomers were immobilized onto a streptavidin-coated BIAcore chip as described under “Experimental Procedures.” Serial dilutions of either sCD8αα wild type or soluble A6 Tax TCR in HBS-EP buffer were flowed over the chip to generate kinetic data. Data were analyzed using BIAeval, Excell, and Origin version 6.1 (Microcal software). K_D values were calculated both by linear Scatchard plots and non-linear analysis assuming 1:1 Langmuir binding (A + B⇆AB) using non-linear curve fitting to the equation AB = B × AB_max/(K_D + B). A, equilibrium binding of sCD8αα interacting with either D227K/T228A HLA A2 (227/8KA), A245V HLA A2, wild type HLA A2 (wildtype), Q115E HLA A2, or A2/K^b α3 domain fusion (A2/Kb) folded around the HTLV-1 Tax epitope. B, summary of sCD8αα/pMHCI affinity measurements obtained for pMHCI monomers bearing different mutations and folded around three different epitopes. DT227/8KA, D227K/T228A. C, the K_D calculated for the interaction between the A6 Tax TCR and either D227K/T228A HLA A2 (DT227/8KA), A245V HLA A2, wild type HLA A2, Q115E HLA A2, or A2/K^b α3 domain fusion folded around the HTLV-1 Tax epitope was shown to be 2.6 ± 0.4, 2.5 ± 0.3, 2.5 ± 0.3, 2.7 ± 0.4, and 2.5 ± 0.2 μm respectively. In all cases indicated errors are S.D. from the mean of three separate experiments. Data shown are from just one representative experiment (hence the values differ slightly from the averages given above).

Fig. 2. The effects of differing pMHCI/CD8 interactions on tetramer staining of human PBMC directly ex vivo

Donor PBMCs were stained with 10, 5, 1, 0.5, and 0.1 μg (with reference to monomer weight) of D227K/T228A HLA A2 (227/8 KA), A245V HLA A2 (245V), wild type HLA A2 (WT), Q115E HLA A2 (Q115E), or A2/K^b α3 domain (A2/Kb) fusion tetramers folded around the CMV pp65-derived peptide epitope NLVPMVATV at 37 °C for 20 min in a final volume of 50 μl. Cells were then washed and stained with anti-CD8 and anti-CD3 monoclonal antibodies for 30 min at 4 °C. Following an additional wash, PBMCs were fixed with 1% paraformaldehyde and analyzed using a FACSCalibur flow cytometer with FlowJo software. At least 300,000 events were collected for each condition. Data plots represent live CD3⁺ lymphocytes. Background MFI values for the CD8⁺tetramer⁻ populations are shown (lower right hand corner of each section); for A2/K^b stains, the MFI value is reported for the entire CD8⁺ T cell population.

Fig. 3. The effects of differing pMHCI/CD8 interactions on tetramer staining of 868 CTL

The CTL line 868 is derived from an HIV-1 infected donor and is specific for the HLA A2-restricted p17 Gag epitope SLYNTVATL. 2 × 10⁵ 868 cells per test were resuspended in 20 μl of PBS and stained with D227K/T228A HLA A2 (277/8KA), A245V HLA A2 (A245V), wild type HLA A2 (wildtype), Q115E HLA A2 (Q115E), or A2/K^b α3 domain fusion (A2/Kb) tetramers folded around the SLYNTVATL peptide at either 0.5, 5 or 50 μg/ml for 20 min at 37 °C. Cells were then stained with CD8-allophycocyanin (clone SK1) for 30 min on ice, washed twice, and then resuspended in PBS. Data were acquired using a FACSCalibur flow cytometer and analyzed using CellQuest software. All HLA A2-SLYNTVATL tetramer-positive cells in the 868 CTL line bear a Vα12-2, Vβ5-6 TCR of identical sequence.

Fig. 4. The effect of the pMHCI/CD8 interaction on TCR/pMHCI dissociation

1.5 × 10⁶ 868 CTL line (A) or 6 × 10⁵ 003 CTL clone (B), 0400 CTL (C), and SLY-10 (D) cells per experiment were resuspended in 100 μl of azide buffer (PBS, 0.1% NaN₃, and 0.5% fetal calf serum) and stained with either A2/K^b-SLYNTVATL (A2/Kb), Q115E HLA A2-SLYNTVATL (Q115E), HLA A2-SLYNTVATL (wildtype), A245V HLA A2-SLYNTVATL (A245V), or D227K/T228A HLA A2-SLYNTVATL (227/8KA) tetramers conjugated to phycoerythrin and 10 μl of 7-amino actinomycin D (ViaProbe; BD Biosciences) for 20 min on ice. For panel E, 6 × 10⁵ of the EBV-specific CTL clone EBV-A were stained with GLCTLVAML versions of the above tetramers. The concentrations of tetramer used were determined previously by titration to give a starting MFI of 200. The 003 clone stains to a similar level with the D227K/T228A tetramer without the need to adjust the concentration (8). Cells were then washed in ice cold azide buffer and resuspended in azide buffer. An excess of unconjugated anti-HLA A2 monoclonal antibody (clone BB7.2; Serotec) was added at 100 μg/ml, and the sample was moved to room temperature to initiate tetramer decay. Samples were taken at 0, 1, 2, 5, 8, 10, 15, 20, 30, 40, and 60 min and analyzed on a FACSCalibur flow cytometer using CellQuest software. Each panel (A–E) shows data from 0–20 min and the least squares fitting of the dissociation curve (percentage of maximal mean fluorescence = background + (100 − background) × exp{−k _off,appt}) from which the apparent off-rate k_off,app values shown in Figs. 5A and 6A were calculated. The apparent off-rate k_off,app is related to the true (single site) off-rate by Equation 4. Cells stained at the same time were kept without anti-HLA A2 blocking antibody and analyzed at 0, 10, 20, 30, 40, and 60 min to demonstrate that tetramers do not decay significantly in the absence of the blocking antibody. An example of this is shown in Fig. S2 of the supplemental data found in the on-line version of this article. The SLY-10 CTL clone did not stain with HLA A*0201 D227K/T228A-SLYNTVATL at any concentration used, so it was not possible to determine an off-rate for the CD8 null tetramer. The lack of data for tetramer stability in the absence of a pMHCI/CD8 interaction makes it impossible to calculate a stabilization factor for this clone or other CTLs with a very low functional avidity. However, comparison to data obtained on the other CTLs suggests that CD8 provides a similar stabilization factor. To date we have examined >20 tetramer-sorted CTL clones, including clones specific for immunodominant viral antigens. All of these clones are of functional low avidity. Such low avidity clones do not stain with CD8 null tetramers (see Ref. for examples). Our experiments show that tetramer staining rapidly induces cell death of CTLs with high functional avidity (Ref. 61). Low avidity CTL grown by tetramer “sort-cloning” are not representative of the types of CTL that function in vivo to clear viral infection. Direct ex vivo staining of CTL specific for the HLA A2 CMV-derived epitope NLVPMVATV shows that these CTL stain well with CD8 null tetramer (Fig. 2). Five further individuals showed a similar pattern (data not shown). CTL specific for the EBV-derived epitope GLCTLVAML can also stain with CD8 null tetramer (data not shown). The decay of the D227K/T228A HLA A2-GLCTLVAML tetramer from EBV-A CTL is shown in the inset of panel E. This CTL clone is of low functional avidity, and it was not possible to stain it to MFI of 200 with this tetramer. The decay shown in the inset, performed at the same time as the other decaying, started at a maximal MFI of 58.

Fig. 5. Modeling the contribution of the pMHCI/CD8 interaction to TCR/pMHCI dissociation of CTL clone 003

A, dissociation constants for pMHCI/CD8 interactions and apparent off-rates for a spectrum of HLA A2 mutants folded around the HIV-1 Gag-specific epitope SLYNTVATL. The apparent off-rate k_off,app was obtained by least squares fitting of the dissociation curve (percentage of maximal mean fluorescence = background + (100 − background) × exp{−k_off,appt}) and is related to the true (single-site) off-rate by Equation 4. DT227/8KA, D227K/T228A; α3K^b, A2/K^b. B, the cube root of the apparent off-rate plotted against the K_D of pMHCI/CD8; the straight line least squares fit confirms the prediction of the Equation 9. C, summary of the stabilization factor and the percentage of maximal stability that can be afforded by the pMHCI/CD8 interaction for each MHCI mutant. DT227/8KA, D227K/T228A.

Fig. 6. Modeling the contribution of the pMHCI/CD8 interaction to TCR/pMHCI dissociation of the CTL line 868

Data as for Fig. 5 but using 868 CTL.