Interaction between the CD8 Coreceptor and Major Histocompatibility Complex Class I Stabilizes T Cell Receptor-Antigen Complexes at the Cell Surface^†

Cell Lines and CTLs

The CTL clone 003 and CTL line 868, both specific for the HLA A2-restricted HIV-1 p17 Gag-derived epitope SLYNTVATL (residues 77–85), have been described previously (31-33). CTLs were grown from cryopreserved stocks in 24-well tissue culture plates in R10 (RPMI medium supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm l-glutamine) containing 5% T-STIM (BD Biosciences) and 200 units/ml proleukin (Chiron) for 2 weeks following re-stimulation as described previously (27). After this time, cells were maintained in R10 plus 25 ng/ml interleukin-15 (Peprotech). Cells maintained in interleukin-15 remained viable and continued to grow for several months without the need for re-stimulation with antigen and/or mixed lymphocyte reaction. CTL clones maintained in interleukin-15 for three months exhibited an activation profile identical in terms of early tyrosine phosphorylation events and effector function (specific lysis of antigen-bearing target cells and production of macrophage inflammatory protein-1β and interferon-γ) to that of CTL maintained in interleukin-2 with periodic re-stimulation. SLYNTVATL-specific 0400 and SLY-10 were similarly maintained. CTL clone EBV-A is specific for the HLA A2-restricted, EBV-derived, BMLFI-encoded epitope GLCTLVAML.

Inclusion Body Preparation

Biotin-tagged HLA A2 heavy chain was expressed under the control of a T7 promoter as insoluble inclusion bodies in Escherichia coli strain BL21(DE3)pLysS (Novagen). Isopropyl-1-thio-β-d-galactopyranoside-induced E. coli were lysed by repeated freeze/thaw cycles to release inclusion bodies that were subsequently purified by washing with a 0.5% Triton X-100 buffer (Sigma) as described previously (34). The D227K/T228A, A245V, and Q115E HLA A2 heavy chain mutants and the A2/K^b α3 domain fusion protein were produced in a similar fashion.

Production of Soluble pMHCI

Soluble biotinylated pMHCI monomers were produced as described previously (35). Briefly, heavy chain and β₂m inclusion body preparations were denatured separately in 8 m urea buffer (Sigma) and mixed at a 1:1 molar ratio. pMHCI was refolded in 2-mercaptoethylamine/cystamine (Sigma) redox buffer with added synthetic peptide (Research Genetics Invitrogen Corp., Huntsville, AL). The HLA A2-restricted peptides used in this study were the HTLV-1 Tax-derived epitope LLFGYPVYV (36), the HIV-derived p17 Gag epitope SLYNTVATL (37), the EBV-derived BMLFI-encoded epitope GLCTLVAML (38), and the CMV-derived pp65 protein epitope NLVPMVATV (39). Following buffer exchange into 10 mm Tris (pH 8.1), the refolded monomers were purified by anion exchange. Purified monomers were biotinylated using d-biotin (Sigma) and BirA enzyme as described previously (34). Excess biotin was removed by gel filtration.

Tetramerization and Flow Cytometry

Fluorescent tetrameric pMHCI complexes were produced by mixing phycoerythrin-conjugated streptavidin (Molecular Probes or Prozyme Inc.) and biotinylated pMHCI monomers at a 1:4 molar ratio, respectively. For ex vivo analysis, 10⁶ peripheral blood mononuclear cells (PBMCs) were stained with the wild type and mutant tetramers shown at the indicated concentrations for 20 min at 37 °C, washed once in fluorescence-activated cell sorter (FACS) buffer (PBS without Ca²⁺/Mg²⁺, 1% bovine serum albumin (w/v), and 0.1% NaN₃), surface-stained with pre-titered allophycocyanin-conjugated anti-CD8 and peridinin chlorophyll protein-conjugated anti-CD3 monoclonal antibodies (BD Biosciences) for 30 min at 4 °C, and then washed twice and fixed in 1% paraformaldehyde. Data were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar Inc., San Carlos, CA). For the in vitro expanded SLYNTVATL-specific CTL line 868, 2 × 10⁵ CTLs were stained as described above with the indicated pMHCI tetramers at the concentrations shown for 20 min at 37 °C, washed, and then stained with allophycocyanin-conjugated anti-CD8 monoclonal antibody (clone SK1; BD Biosciences) and 7-amino actinomycin D (ViaProbe; BD Biosciences) for 30 min on ice prior to two further washes and data collection; analysis was performed with CellQuest software (BD Biosciences). All pMHCI tetramers used in this study were made fresh for the week of use from pMHCI monomers stored at −80 °C to minimize the effect of stability differences (15). Once prepared, tetramers were stored in the dark at 4 °C.

pMHCI Tetramer Decay Assay

Indicated numbers of CTL were stained in 100 μl of azide buffer (PBS, 0.1% NaN₃, and 0.5% fetal calf serum) for 20 min on ice with a concentration of tetramer, previously determined by titration, that gave a starting mean fluorescence intensity (MFI) of 200; 7-amino actinomycin D (ViaProbe, BD Biosciences) was included so that dead cells could be gated out of the analysis. After washing twice in ice-cold azide buffer, CTLs were resuspended in azide buffer, split into two separate aliquots, and placed at room temperature. To one sample, an excess of unconjugated anti-HLA A2 monoclonal antibody (clone BB7.2; Serotec) at 100 μg/ml was added to block tetramer rebinding. Cells were then taken at time points 0, 1, 2, 5, 8, 10, 15, 20, 30, 40, and 60 min, resuspended in PBS, and analyzed on a FACSCalibur flow cytometer. The remaining sample was left without BB7.2, and controls were analyzed at the 0-, 10-, 20-, 30-, 40-, and 60-min time points. Decays were repeated for all mutant tetramers in the panel on the same day. Parameter validation for decay assays is shown in Figs. S1 and S2 of the supplemental data, available in the on-line version of this article.

Biophysical Validation of Mutant pMHCI Affinities for CD8 and TCR

The D227K/T228A mutation in the α3 domain of HLA A2 has been shown previously to abrogate CD8 binding (8); the A245V mutation reduces CD8 binding by >4-fold (16). The biophysical properties of the Q115E α2 domain mutant and the A2/K^b hybrid pMHCI proteins were determined by SPR using a BIAcore 3000™ (BIAcore AB, St. Albans, UK) machine. sCD8αα wild type was prepared as described previously (40). The A6 TCR specific for the HLA A*0201-restricted HTLV-1 Tax epitope LLFGYPVYV was refolded as described previously (41). All proteins for analysis were diluted into HBS-EP buffer (BIAcore AB) containing 10 mm HEPES (pH 7.4), 150 nm NaCl, 3.4 mm EDTA, and 0.005% surfactant P20. A standard amine coupling kit (BIAcore AB) was used to activate the surface of a research grade CM5 sensor chip (BIAcore AB). Streptavidin was covalently coupled to the chip surface by primary amines through the injection of a 0.2 mg/ml streptavidin solution (Sigma) diluted in 10 mm sodium acetate (pH 4.5) over the surface. Biotinylated pMHCI monomers were immobilized onto the chip surface at ∼1,000 response units in each flow cell. 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). Each batch of protein was validated by SPR prior to experimentation.

Analysis of Tetramer Decay

We assume the following kinetics for x₁, x₂, and x₃, which represent the surface densities of monovalently bound, bivalently bound, and trivalently bound tetramers, respectively, as shown in Equations 1-3,

where the prime indicates the derivative with respect to time, μ is the single-site rebinding rate, and k_off is the single-site off-rate. Because tetramers do not rebind to CTL (Fig. S1 in the supplemental data), we can safely assume that tetramers in solution are captured by antibody before rebinding can occur. If rebinding is slow (relative to k_off) the dissociation curve is sigmoid, with a shape governed by the mixture of monovalently, bivalently, and trivalently bound forms present at the onset of the dissociation experiment with an exponential tail of time scale 1/k_off. On the other hand, if rebinding is fast the ratios x₁:x₂ and x₂:x₃ rapidly attain a quasi-equilibrium (irrespective of the initial mixture), and the dissociation curve approaches simple exponential decay with a much reduced relaxation rate 3k_off³/μ². Because our data exhibit exponential decay (Fig. 4), we can confidently assume that our experimental system is in this rapid rebinding regime. (It is notable that the sigmoid curves and exponential curves arise as special cases of a single theory.) As shown here,

Equation 4 relates the apparent off-rate obtained from non-linear least-squares regression on the dissociation curve, k_off,app, to the true (single-site) off-rate. The proportionality constant (μ²/3)^⅓ cannot be determined from our data. However, our conclusions depend on ratios of off-rates, which are readily obtained as the cube roots of ratios of the apparent off-rates.

Dependence of the TCR/pMHCI Off-rate on CD8

Let the TCR/pMHCI off-rate be denoted by k_off° when the MHC molecule is CD8-bound and by k_off* when MHC is not bound to CD8. We assume that these two rates do not depend on the affinity of the MHCI/CD8 interaction. Furthermore, let γ denote the rate at which TCR-bound MHCI molecules associate with CD8; γ is proportional to the surface density of unoccupied (free) CD8 molecules on the T cell. Finally, let ρ denote the rate at which MHCI and CD8 dissociate. Then we attain Equation 5,

where [CD8]_F is the surface density of free CD8 molecules in the contact area and K_D,2D is the two-dimensional dissociation constant of the MHCI/CD8 interaction. We now develop a simple mathematical model that describes how the true single-site off-rate k_off is derived from k_off° and k_off*. Consider a pMHCI molecule that engages a TCR molecule at time t = 0. Let P°(t) denote the probability that dissociation of this TCR/pMHCI does not occur before time t while the MHCI is not associated with CD8 at time t, and let P*(t) denote the probability that TCR/pMHCI dissociation occurs after time t while the MHCI is associated with CD8 at time t. Thus, P°(t) + P*(t) is the probability that TCR/pMHCI dissociation occurs later than t; hence, k_off equals the rate of change of −ln{P°(t) + P*(t)}. The rate of change of P° + P* equals the following,

as a routine argument shows. Using this equation and the fact that MHCI/CD8 kinetics are much more rapid than the TCR/pMHCI kinetics (12), we obtain P*/(P* + P°) = γ/(ρ + γ), which can be used to find the decay rate of P° + P* as shown here,

which we can rewrite as this,

by using Equation 5 and κ = [CD8]_F K_D,3D/K_D,2D, where K_D,3D denotes the three-dimensional dissociation constant of the MHCI/CD8 interaction. Combining this equation with Equation 4 and κ ≫ K_D,3D, an assumption warranted by our data, we obtain Equation 9,

which is the linear relationship shown in Figs. 5B and 6B.

The Design of Mutations to Increase the Interaction between pMHCI and CD8

The D227K/T228A mutation in the α3 domain of HLA A2 abrogates CD8 binding (8); the A245V mutation reduces CD8 binding by >4-fold (16). To develop similar HLA A2-based reagents with enhanced binding affinities for CD8αα, we employed the multi-scale approach used by Glick et al. (42, 43). The algorithm was limited to four feature points. The surface of the CD8αα protein that forms contacts with HLA A2 (3) was systematically searched using amino acid side chains as “probes.” A Gln-115 to Glu mutation in the α2 domain was selected for further molecular dynamics study. The wild type HLA A2 Q115 Oε1 atom forms a weak H-bond interaction with the CD8α1 R4Nη1 atom. (The Oε1 … Nη1 distance in the crystal structure is 3.18Å). This interaction was replaced by a shorter H-bond between HLA A2 Q115E Oε1 and CD8α1 R4 Nη1 atoms; the molecular dynamics simulation showed that the Oε1 … Nη1 average distance was 2.56Å and fluctuated from 2.42 to 2.82 Å. This short distance also indicates that the HLA A2 Q115E carboxylate and CD8α1 R4 guanidinium moieties form a strong electrostatic interaction that is likely to increase the affinity of binding between the two biomolecules. SPR studies supported these predictions (Fig. 1). The human/murine hybrid HLA A2/H2-K^b α3 domain fusion protein (A2/K^b), which has a substantially increased binding affinity for human CD8αα, has been described previously (44).

Verification That Mutations Affect the pMHCI/CD8 Interaction without Altering the TCR/pMHCI Interaction

SPR was used to determine that the K_D values of the pMHCI/CD8 interaction for D227K/T228A HLA A2, A245V HLA A2, wild type HLA A2, Q115E HLA A2, and A2/K^b folded around the HTLV-1 epitope (LLFGYPVYV) were >10,000, 498, 137, 98, and 10.9 μm respectively (Fig. 1A). The K_D values of the pMHCI/CD8 interaction for D227K/T228A HLA A2, wild type HLA A2, Q115E HLA A2, and A2/K^b folded around the HIV-1 epitope SLYNTVATL were shown to be >10,000, 128, 87, and 9 μm, respectively (Fig. 1B). These substitutions in the α3 or α2 domain of the pMHCI molecule did not affect TCR binding (Fig. 1C). This spectrum of HLA A2 molecules that have normal TCR/pMHCI interactions but a range of pMHCI/CD8 interactions exceeding 1,000-fold was subsequently used to study the role of the pMHCI/CD8 interaction in the binding of pMHCI antigen.

The CD8 Interaction Determines the Pattern of pMHCI Tetramer Staining at Both Subnormal and Supranormal Affinities

Multimeric pMHC molecules have recently revolutionized T cell immunology by enabling the direct visualization, enumeration, phenotyping, and sorting of T cells based on the antigen specificity of their TCRs (45-47). Emerging evidence suggests that the use of multimerized pMHCI antigens with decreased CD8 binding affinities enhances the specificity of these reagents in relation to background staining (48) and can be used to identify CTLs with a high sensitivity for antigen (49, 50). We used our panel of pMHCI tetrameric complexes with differing affinities for CD8 to examine the staining of human CD8⁺ T cells specific for the HLA A2-restricted CMV pp65-derived epitope NLVPMVATV across a range of concentrations directly ex vivo (Fig. 2). These data show the utility of these reagents for staining direct ex vivo human PBMCs and eliminate the possibility that the mutations we have engineered into HLA A2 to alter CD8 binding inadvertently introduce new binding properties into the MHC class I molecule. A distinct antigen-specific population was identified with the corresponding pMHCI tetramers of Q115E HLA A2, wild type HLA A2, A245V HLA A2, and D227K/T228A HLA A2; the magnitude of the detected population was equivalent for all of these reagents across all concentrations tested (Fig. 2). Similar results were obtained with PBMCs from two other donors with these CMV-specific reagents and in two further donors with pMHCI tetramers of wild type HLA A2, A245V HLA A2, and D227K/T228A HLA A2 folded around the EBV BMLFI-derived peptide GLCTLVAML (data not shown). Staining intensity declined with decreasing concentrations in all cases as expected. The most notable differentiating feature was background staining. As CD8 affinity increased, the CD8⁺tetramer⁻ population shifted proportionately toward the CD8⁺tetramer⁺ population, as demonstrated by the mean fluorescence intensities of the CD8⁺tetramer⁻ subset. This trend extended to the extreme case in which all CD8⁺ T cells were stained by the A2/K^b tetrameric reagent (Fig. 2). Importantly, direct ex vivo staining of PBMC with CMV-specific tetramers showed that wild type and CD8 null reagents could identify a similar population of antigen-specific cells (Fig. 2). This finding suggests that these T cells bear a TCR with a relatively high affinity for cognate antigen (49).

The pMHCI/CD8 Interaction Affects pMHCI Tetramer Dissociation

The analysis of interaction kinetics between pMHCI and cell surface TCR has been hindered by the extremely short half-lives of such interactions (1–12 s at 25 °C). Tetrameric pMHCI molecules have considerably longer interaction times and allow a quantitative assessment of dissociation. We examined the decay of pMHCI tetramers with altered pMHCI/CD8 interactions but unaltered TCR/pMHCI interactions from the cell surface of an azide-poisoned CTL line that recognizes the HLA A2-restricted, HIV-1 p17 Gag-derived epitope SLYNTVATL. This epitope is frequently immunodominant in HLA A2⁺ HIV-infected patients, and the prevalent CTLs in line 868 have been shown previously to be expanded in the patient from whom the line was derived (32). The corresponding pMHCI proteins of A2/K^b, Q115E HLA A2, wild type HLA A2, A245V HLA A2, and D227K/T228A HLA A2 exhibit identical TCR binding but have different CD8 binding (8, 15, 16, 49) (Fig. 1B). All of these reagents stained a population of cells in the 868 CTL line (Fig. 3). The A2/K^b reagent stained all CD8⁺ cells when used at high concentrations (Fig. 3). This result is consistent with our finding that this reagent stains all CD3⁺CD8⁺ human peripheral blood mononuclear cells regardless of their TCR specificity (49) (Fig. 2). When tetrameric A2/K^b is used at a low concentration, the cells with a TCR specific for the SLYNTVATL peptide appeared to out-compete other CD8⁺ cells for this reagent (Fig. 3; left column). Staining intensity with the D227K/T228A reagent was almost three times lower than with the wild type reagent when 0.5 μg/ml pMHCI was used (Fig. 3), consistent with the 2.5-fold difference in MFI we observed previously with this CTL line (8). Thus, these CTLs exhibit some dependence on the pMHCI/CD8 interaction for staining with pMHCI tetramers in contrast to the 003 CTL clone that recognizes the same antigen (8); these observations suggest that 003 CTL may bear a TCR with higher affinity for the cognate ligand and indicate that there is some variability between SLYNTVATL-specific CTLs in their requirements for the pMHCI/CD8 interaction to aid the binding of pMHCI tetramers.

The decay of pMHCI tetramers from the cell surface of 868 CTLs clearly correlates with the affinity of the pMHCI/CD8 interaction (Fig. 4A). To study this finding further, we examined CTL clone 003, which expresses a different SLYNTVATL-specific TCR than 868 CTL and is derived from a different patient (31, 32). This clone is believed to be the dominant clone that recognized this epitope in vivo (32). We have shown previously that wild type and D227K/T228A pMHCI tetramers exhibit a similar on-rate and stain these cells within 30 s (8). Using our panel of tetramers with altered CD8 binding properties, we confirmed that a decreased pMHCI/CD8 interaction leads to more rapid tetramer dissociation and that an increased pMHCI/CD8 interaction results in slower tetramer dissociation (Fig. 4B). Similar data were obtained with three further sets of CTLs (Fig. 4, C-E). Thus, the pMHCI/CD8 interaction affects the stability of the TCR/pMHCI interaction on the cell surface. The decay of pMHCI tetramers from 003 CTL (Fig. 4B) was slower than for 868 CTL (Fig. 4A). The former CTL might have a higher affinity TCR, consistent with our finding that 003 CTL are less dependent on CD8 for tetramer binding (8) than 868 CTL (Fig. 3).

The pMHCI/CD8 Interaction Stabilizes the TCR/pMHCI Interaction ∼2-Fold

The apparent off-rates in the tetramer dissociation experiments (Figs. 4-6) exhibit a wide variation; the D227K/T228A HLA A2 decay rate differs from that of wild type HLA A2 by a factor of ∼10. However, these differences in apparent off-rates do not translate directly into single-site off-rates, because tetramer kinetics involves TCR-bound forms of three different valancies (see Equations 1-3). In the regime where tetramer decay curves are virtually exponential, the true ratio of off-rates is the cube root of the ratio of apparent off-rates. In particular, the tetramer dissociation curve fit for D227K/T228A HLA A2 from 003 CTL indicates that (3/μ²)^⅓k_off° = 0.859 min^−⅓ (see Experimental Procedures for notation). The intercept of the straight line fit (Equation 9) shown in Fig. 5B yields (3/μ²)^⅓k_off* = 0.398 min^−⅓. These data suggest that the maximum effect of the pMHCI/CD8 interaction is a prolongation by a factor of 2.16 of the mean TCR/pMHCI binding time. The fit also indicates that (3/μ²)^⅓ k_off°/κ = 0.29 min^−⅓/nm, whence κ = 3 mm, which, in the interaction between a T cell and an antigen-presenting cell, would translate as 6 × 10⁴ free CD8 molecules in the contact area between a T cell and an antigen-presenting cell if we assume σA = 30 × 10⁻²¹ m³, where A is the area of the contact interface between the cells and σ is the “confinement length”; the free CD8 count equals Aσκ. Taken together, the tetramer decay experiments indicate that the pMHCI/CD8 interaction stabilizes by ∼2-fold the interaction between the TCR of HIV Gag-specific clone 003 and HLA A2/K^b-SLYNTVATL. The actual effect depends on the fraction of time spent by the MHC molecule in the CD8-bound form, and this fraction increases with increasing affinity. Thus, the wild type pMHCI/CD8 induces a stabilization factor of 1.95, which is close to the maximum of 2.16 by virtue of its low K_D of 128 μm. A similar CD8-stabilization effect was observed for 868 CTLs (Fig. 6) and other CTLs with different functional sensitivities (summarized in Table I). We find that pMHCI tetramers rapidly induce the cell death of so-called “high avidity” CTLs (61).² This may also be true when these reagents are used to sort-clone CTL. The failure of CTL clones with low functional avidity/sensitivity to stain with CD8 null tetramers (44) (Fig. 4)² makes it impossible to calculate an apparent off-rate for these reagents from these clones. The lack of stability in the absence of a pMHCI/CD8 interaction precludes calculation of a CD8-mediated stabilization factor for such CTLs. Nevertheless, comparison of the off-rates of the other tetramers from the surface of CTL clone SLY-10 to those of other CTLs (Fig. 4) strongly suggests that the pMHCI/CD8 interaction provides a similar stabilization factor of ∼2-fold for CTL clones of very low functional avidity.