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for MHC Class I Reveals Mutants with Enhanced Binding1
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* Department of Laboratory Medicine,
Section of Immunobiology, and
Department of Genetics, Yale University, New Haven, CT 06520
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Abstract |
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heterodimer acts as a coreceptor with the TCR, to enhance sensitivity of the T cells to peptide/MHC class I, and is two orders of magnitude more efficient as a coreceptor than the CD8. To understand the important interaction between CD8 and MHC class I, we created a panel of CD8 mutants and identified mutations in the CDR1, CDR2, and CDR3 loops that decreased binding to MHC class I tetramers as well as mutations that enhanced binding. We tested the coreceptor function of a subset of reducing and enhancing mutants using a T cell hybridoma and found similar reducing and enhancing effects. CD8-enhancing mutants could be useful for immunotherapy by transduction into T cells to enhance T cell responses against weak Ags such as those expressed by tumors. We also addressed the question of the orientation of CD8 with MHC class I using CD8 mutants expressed as a heterodimer with wild-type CD8 or CD8. The partial rescuing of binding with wild-type CD8 compared with wild-type CD8 is consistent with models in which either the topology of CD8 and CD8 binding to MHC class I is different or CD8 is capable of binding in both the T cell membrane proximal and distal positions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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homodimers and CD8 heterodimers. On the majority of MHC class I-restricted conventional T cells and thymocytes, the predominant form of CD8 expressed is the CD8 heterodimer and there is experimental data suggesting that regulation of CD8 levels on the T cell surface, particularly CD8, may be a means for CD8 T cells to regulate their effector function (1, 2). Interaction of CD8 with its MHC I ligand is known to decrease the threshold for peptide-MHC class I (pMHC I)3 TCR interactions required for CTL activation and significantly increase the cytotoxic response, particularly in the recognition of peptides with low affinity for the TCR (3). The CD8 homodimer form of CD8 is not concordant with MHC class I restriction of the TCR and can be expressed on different cell lineages including dendritic cells (4), 
intestinal intraepithelial lymphocytes (5), and subsets of NK cells (6). Although both soluble CD8 and CD8 forms have been shown to have similar affinity for classical MHC class I by surface plasmon resonance studies (7), CD8 is a much better T cell coreceptor and it has been suggested that CD8 may mediate different functions from that of CD8, in particular due to its high affinity for the nonclassical class I molecule TL (8).
Several studies have provided evidence of how CD8 contributes to T cell recognition and response to cells expressing foreign Ags. Although the cytoplasmic domain of the CD8 chain contains the docking site for p56lck required for the initiation of early T cell signaling (9, 10), it is palmitoylation of the CD8 chain that facilitates the partitioning of CD8 into lipid rafts which is critical for the ability of CD8 to act as a coreceptor with the TCR (11). A direct association of CD8 with the TCR was suggested by cocapping studies, which revealed CD8/TCR interactions after capping with anti-CD8 Abs but not anti-CD8 Abs (12). Subsequently, a direct association between CD8 and CD3 was reported by Doucey et al. (13) using both thymocytes and a T cell hybridoma. The association of CD8 with CD3 contributes to bringing the TCR into lipid rafts and it has been suggested that the CD8-pMHC interaction increases the probability of an encounter of pMHC with the appropriate TCR (14). In support of this hypothesis, an interesting study by Yachi et al. (15) found that noncognate CD8-pMHC I interaction can enhance cognate Ag recognition perhaps by concentrating pMHC and CD8 to the immunological synapse.
The precise details of the CD8 binding to MHC class I and in particular, the contribution of CD8 to the interaction of CD8 with MHC class I, are not known. During the course of this study, the crystal structure of the Ig domains of CD8 was published showing that CD8 is structurally very similar to CD8 (16). The CDR3 loops which are at the center of the MHC class I-binding interface share the most similarity, indicating that CD8 and CD8 dimers likely bind to MHC class I similarly. However, there are some potentially important differences. The CDR1 loop of CD8 tilts away from the CDR2 and CDR3 loops which may decrease its contribution to MHC class I binding, whereas the CDR2 loop tilts closer to the CDR3 loop which may result in greater interaction with the CD loop on MHC class I. The same study used a mutational approach and identified mutations in the CDR2 and CDR3 loops of CD8 that decreased coreceptor function.
To better understand how CD8 interacts with MHC class I, we created a large panel of mutants to identify residues of CD8 involved in binding to MHC class I using assays to measure both MHC class I binding and T cell activation. We identified residues in the CDR1, CDR2, and CDR3 loops that decreased MHC class I binding and T cell activation. We also identified mutations that enhanced binding to MHC class I tetramers. Because CD8 increases the avidity of pMHC/TCR interactions, we wanted to address the possibility that enhanced CD8-MHC class I binding correlated with improved coreceptor function as this could provide a means of enhancing the T cell response to low-affinity Ags such as those expressed by tumor cells. We show that activation of a T cell hybridoma was improved in cells expressing CD8 mutants that enhanced binding to MHC class I.
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Materials and Methods |
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Mutagenesis of solvent-exposed residues on mouse and human CD8 was performed using the Quickchange Mutagenesis kit from Stratagene. Oligos were designed to change the designated amino acid and to create a restriction site to identify mutants (Table I). Mutagenesis was conducted in the expression vector (pCDL-SR296) and each construct was sequenced to confirm the presence of the mutation.
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CD8 expression on COS-7 cells
COS-7 fibroblasts were cultured and maintained in RPMI 1640 with 10% FCS (HyClone). Eighteen to 24 h before transfection, cells were seeded at a concentration of 4 x 105 cells/60-mm dish. Cells were transfected using Lipofectamine 2000 (Invitrogen Life Technologies). With mouse CD8, to ensure predominant expression of CD8 heterodimers, we used 1 µg of CD8 and 2 µg of CD8 cDNA for transfection. For human CD8, because CD8 can come to the surface by itself (17), we used equal amounts of each cDNA (total 3 µg). DNA was diluted in 0.5 ml of Optimem and mixed with 0.5 ml of Optimem containing 8 µl of Lipofectamine 2000. Reagent mixtures were incubated in the dark at room temperature for 15 min, followed by addition of 1 ml of Optimem and layering onto the COS-7 cells. Five hours posttransfection, the reagent mix was removed and 4 ml of culture medium was added to each plate. Cells were given fresh medium every 24 h and harvested for phenotypic analysis at 48 h.
Preparation of tetramers
OVAp or SIYp H-2Kb tetramers were prepared with peptides SIINFEKL or SIYRYYGL, respectively, and HLA-A2 tetramers were prepared with the EBV peptide GLCTLVAML using the previously described method (18). Briefly, human 2-microglobulin and a truncated form of either H-2Kb H chain or HLA-A2, in which the transmembrane and cytosolic domain had been removed and a specific biotinylation site was added to the C terminus (19), were expressed in Escherichia coli strain BL21 (DE3) LysS. Inclusion bodies were purified and the proteins were refolded as described previously (20). Refolding was performed at 10°C in the presence of 25 µg/ml peptide (Research Genetics) and protease inhibitors: pepstatin A (1 µg/ml), leupeptin (1 µg/ml), and PMSF (0.4 mM). Soluble monomeric complexes were purified by gel filtration over a Superdex 200HR column (Amersham Biosciences) and enzymatically biotinylated by overnight incubation with purified BirA at room temperature with components as follows: 5 µM HLA-A2/peptide, BirA enzyme (1.5 x 106 U; Avidity), 80 µM biotin, 10 mM ATP, 10 mM MgOAc, and 20 mM bicine. Unbound biotin was removed by gel filtration and the purified monomers were tetramerized by incubation with PE-labeled streptavidin (Molecular Probes) at a molar ratio of 4:1.
FACS analysis of CD8 expression and tetramer binding
Mouse CD8 expression was determined by staining with mAbs CT-CD8-FITC (Caltag Laboratories) and 53.6.7-FITC (BD Pharmingen) before tetramer binding assays. In addition, CD8 expression was confirmed by staining with CD8 Abs CT-CD8-PE (Caltag Laboratories), KT112 and H35.17 (gift from S. Jameson, University of Minnesota, Minneapolis, MN). For analysis of MHC tetramer binding, 1 µl of H2Kb tetramer was added to a minimum of 105 cells and incubated for 1 h on ice on a rocker platform. Following incubation, cells were washed twice in buffer (PBS with 1% FCS and 0.1 mM sodium azide), then analyzed on a FACScan or FACSCalibur flow cytometer.
Human CD8 expression was assessed using the CD8 Ab OKT8, and the CD8 Abs 5F2 (Santa Cruz Biotechnology) and 2ST8 (Beckman Coulter). For analysis of MHC class I binding, 1 µl of HLA-A2 EBV tetramer was added to cells for 1 h on ice. For each experiment, the COS-7 cells were transfected with the vector plasmid alone. Staining of these cells with Ab or tetramer served as a baseline fluorescence. Results for each mutant were calculated using both the percent-positive cells relative to vector alone and the mean fluorescence of the positive population. The relative expression level was also taken into consideration, using the following formulas: ((MFI tetramer binding)/(MFI Ab binding)) x ((percent positive tetramer – tetramer binding to vector)/(percent CD8 Ab – Ab binding to vector)) = binding index (BI); MFI is the mean fluorescence intensity.
The BI of tetramer binding to CD8 wild type was taken as 100% and results expressed relative to wild type: (BI mutant x 100)/(BI wild type) = percent binding relative to wild type.
IL-2 production by 2C T cell hybridomas expressing CD8 mutants
2C-TCR hybridoma cells expressing CD8 were provided by D. Kranz (University of Illinois, Urbana, IL). CD8 wild-type and mutant cDNAs were cloned into the retroviral expression vector pBMN and transduced into T cells as previously described (21). Cells were sorted to ensure that levels of CD8, CD8, and CD3 were comparable. To measure T cell activity, T cells (1 x 105) were incubated with RMA-Kb cells (1 x 105) preloaded (30–60 min) with various concentrations of peptide (either the SIYRYYGL peptide recognized by the 2C TCR or the OVA peptide SIINFEKL as a negative control) in 96-well U-bottom plates at 37°C and 5% CO2. Supernatants were harvested after 24 h and assayed for IL-2 by ELISA.
ELISA
ELISA was performed using the anti-mouse IL-2 Ab JES6-1A12 (Biolegend) as the capture Ab. Plates were blocked for 2 h with 5% BSA in PBS. Cell supernatants were added and incubated for 2 h followed by IL-2 detection using biotinylated JES-54H followed by streptavidin-HRP and developed using TMB substrate (Biolegend). Mouse rIL-2 was used to create a standard curve.
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Results |
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mutations on MHC class I binding
There is 
20% homology between the Ig domains of CD8 and CBD8, and the majority of the structural information we have on the CD8-MHC class I interaction has come from studies on CD8 homodimer binding to class I. Assuming CD8 binds in a similar manner, we mutated residues in the CDR-like loops and/or highly conserved amino acids in CD8 to determine whether they were involved in MHC binding. We transiently expressed these mutants with wild-type CD8 in COS-7 cells and analyzed their ability to bind MHC class I tetramers. There was a decrease in tetramer binding to cells expressing CD8 with mutations of residues in the CDR1, CDR2, and CDR3 loops of CD8 (Fig. 1A). In the CDR1 loop, mutant K23A had no effect whereas mutation to a negatively charged amino acid (K23D) did decrease MHC class I binding. It is possible that we did not see any effect with the alanine substitution because other side chains compensated for this change by a structural rearrangement of side chains, whereas the charged aspartic acid disrupted the binding interface. The observed decrease could also be a result of a structural effect due to the instability caused by an unpaired negatively charges residue. In the CDR2 loop, mutation of K55D significantly decreased MHC class I binding and in the CDR3 loop, mutants V99R, S101A, and K103D all decreased MHC class I tetramer binding.
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. We mutated five lysines (K130,132–135), three of which are conserved by charge between species (130, 134, 135). Mutation of these lysines to alanine had no effect on tetramer binding.
In addition to mutations that decreased MHC class I tetramer binding, we also identified mutations that increased binding. These mutations were either in the CDR1 loop, I25A, the CDR2 loop, S53L, S54V, or a strand, L58R. A double mutant of I25A/L58R showed greatly enhanced binding of MHC class I tetramers (300% relative to wild type). Examples of MHC tetramer staining to COS-7 cells expressing wild-type or mutant forms of CD8 that enhance or decrease binding are shown in Fig. 1B.
To determine whether the contacts for human CD8 binding to MHC class I were the same as in mouse, we created a panel of human CD8 mutants and used human MHC class I tetramers (HLA-A2) to determine the effects on binding. We focused on mutating human CD8 residues that were analogous to mouse CD8 that had shown an effect on MHC class I tetramer binding (Fig. 2A). A number of key contact residues are conserved between human and mouse. For murine residues that demonstrated decreased binding to MHC tetramer when mutated, the equivalent human residues when mutated, exhibited a similar effect. In the CDR1 loop, K23D decreased tetramer binding (equivalent to murine K23), in the CDR2 loop K56D (equivalent of mouse K55) decreased tetramer binding, and in the CDR3 loop S100A (equivalent of mouse S101) resulted in decreased MHC class I binding (Fig. 2B). The analogous mutations that had greatly increased MHC class I binding in the mouse (I24A, S54V, and I59R) resulted in a very modest increase in binding to human MHC class I.
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Abs
Mutagenesis of both mouse and human CD8 allowed the identification of the epitopes of several Abs. In mouse CD8, mutation of K55 reduced the binding of H35.17. This mutation also decreased MHC class I binding, which explains the finding that this Ab can block MHC class I binding by 90% (Fig. 3).
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mutation of I59 destroyed binding of 5F2 and mutation of S100, a contact site for MHC class I, showed a decrease in the CD8 Ab 2ST8 (Fig. 4). 2ST8 was previously shown to bind to an epitope formed by the CD8 heterodimer (17). This Ab significantly blocked MHC class I binding (
65%) which is consistent with the epitope mapping to the CDR3 loops. Pretreatment of cells with 5F2 also reduced tetramer binding by 35%.
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mutations on T cell activation
To determine the effect of some of these mutations on T cell coreceptor activity, we introduced CD8 mutants into a T cell hybridoma that expressed the 2C TCR and CD8. We were particularly interested in determining whether the increased MHC class I binding we observed with some CD8 mutants would result in enhanced T cell activation. We used different concentrations of the 2C peptide SIYRYYGL to stimulate T cell hybridomas and measured IL-2 production as a marker for T cell activation. The levels of CD8 and CD3 and were comparable in all hybridomas tested (Fig. 5A). In addition, we stimulated each transfectant with PMA and ionomycin to demonstrate that they were still capable of making equivalent IL-2 responses by this stimulation. This was indeed the case as shown in Fig. 5B. Fig. 5C show that mutants I25A/L58R and S53L (i and ii, respectively) increased IL-2 production at all peptide concentrations and the cells could respond to lower concentrations of peptide. The enhanced T cell coreceptor response was still Ag specific, because 2C hybridomas expressing the enhancing mutants did not respond to the negative control peptide SIINFEKL which can bind to H-2Kb but is not recognized by the 2C TCR.
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alone.
Topology of CD8 binding to MHC class I
The cocrystal structure of CD8 homodimer with MHC class I demonstrated that the binding of CD8 was asymmetrical with 70% of the contact area coming from the membrane proximal CD8a-1 subunit (22, 23). An important question in CD8 binding to MHC class I is whether CD8 preferentially binds in the T cell membrane proximal (CD8a-1) or membrane distal (CD8a-2) position.
We compared the effect of several CD8 mutants on MHC class I binding when expressed as CD8 homodimers and CD8 heterodimers. The CD8 mutants were located at position R8 or S31, residues that made contact only in the CD8a-1 membrane proximal subunit or position K62, that only made contact in the CD8-a2 distal subunit (23). We hypothesized that the ability of CD8 to rescue the effect of the CD8 mutation would provide information regarding the orientation of CD8 in the binding of CD8 binding to MHC class I. If CD8 bound to MHC class I with a similar topology to CD8, then if CD8 was in the membrane proximal position, it would rescue the effects of the R8 and S31 mutations whereas if it was in the membrane distal position, it would rescue the effect of the K62 mutation. We did observe CD8 mutants which showed differences in binding to the MHC tetramer when expressed in the homodimer vs heterodimer form (Fig. 6A). For example, R8 and S31, the CD8a-1 only contact residues, showed partial rescue in binding when CD8 is coexpressed with these mutants (Fig. 7 shows the location of these residues in both CD8 homo- and heterodimer binding to MHC class I).
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is binding in the CD8a-1 position and we do not see complete rescue due to the presence of mutant CD8 homodimers. Because an Ab against CD8 showed almost complete blocking of tetramer binding (Fig. 3), this would indicate that almost all of the CD8 expressed is in the heterodimer form. However, to formally test whether CD8 homodimers were preventing a complete rescue, we did a titration experiment with different ratios of CD8:CD8. The rescue effect of CD8 was greatest when CD8 was in excess but we never obtained >30% rescue (Fig. 6B). In contrast, if we looked at tetramer binding to CD8 wild-type and CD8 mutant (S31A) at different ratios, we could see rescue of 70% when the ratio was 2:1 WT:MT. If CD8 only bound in the CD8a-1 position, we would expect 100% rescue whereas if CD8 bound in only the membrane CD8a-2 we would expect no rescue. The partial rescue observed suggests that either CD8 and CD8 do not bind to MHC class I with the same topology or that CD8 is capable of binding to MHC class I in two different orientations. The fact that the K62A mutation had 50% decreased binding in the heterodimer form as compared with the homodimer forms also supports our hypothesis that the CD8-MHC class I interaction is not a simple replacement of the Ig domain for the Ig domain in the CD8-MHC class I interaction in a membrane proximal position.
Though CD8 residues K12 and K13 are not contact residues in the CD8-MHC class I cocrystal structure (23), certain mutations in those residues affected binding to MHC class I. CD8 mutant K12EK13E also showed different effects on MHC binding when expressed as either CD8 homodimers or CD8 heterodimers (both single and double mutants). We observed that the effect of mutation from lysine to alanine was not rescued by CD8, whereas there was rescue with the lysine to glutamic acid change. We previously showed that these mutations did not alter binding of TL (24), therefore there is no gross conformational change induced by these mutations. The K12/K13 residues may be affecting MHC class I binding in the CD8a-1 domain because of their proximity to the 1/2 domain of MHC class I. If CD8 bound in the membrane proximal a-1 position, the effect of these mutations on MHC tetramer binding would be less which is what we observed. But again the rescue was not 100%.
We modeled the location of CD8 mutants that had an effect on MHC class I binding with CD8 in both membrane proximal and distal positions (Fig. 8). For all mutants shown to alter MHC class I binding (either enhance or reduce), the residues appear to be in a better position to contact MHC class I when CD8 is in the membrane proximal (CD8a-1) position for CD8. However, the extent of similarity of the interaction between CD8 and CD8 with MHC class I is yet to be determined, and as mentioned previously, it is possible that CD8 can bind in either position or with altered topology.
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Discussion |
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for T cell coreceptor function. Therefore, a better understanding, at the molecular level regarding how CD8 interacts with MHC class I is an important question. We have created an extensive panel of CD8 mutants and tested them in both a direct MHC class I-binding assay and in a T cell activation assay. One of the most interesting findings from our study is the identification of mutations that cause a significant increase in CD8 binding to MHC class I which resulted in enhanced T cell activation in a hybridoma T cell line. It remains to be determined how the CD8-enhancing mutants are exerting their effect, however, the models we have generated suggest that additional contacts with the negatively charged C-D look in the 3 domain of MHC class I or with 2-microglobulin may contribute to enhanced binding. We identified residues in all three CDR loops that decreased MHC class I binding, whereas in the study by Chang et al. (16), they identified mutations in the CDR2 and CDR3 loops that decreased IL-2 production in a T cell hybridoma, but not in the CDR1 loop. The reduced binding of the K23D mutant, together with the enhancing effect of I25A, suggests that the CDR1 loop does contribute to the binding with MHC class I.
Some of our results confirmed the previously published contact residues (16). In the CDR2 loop, K55 is a contact residue as mutation to aspartic acid or alanine can reduce MHC class I binding and/or T cell activation and in the CDR3 loop S101 and K103 are confirmed contact residues. In addition in the CDR3 loop, we identified V99 as a key contact residue. Interestingly, as with K23, we did not see an effect with K103A, but did observe a decrease in binding when K103 was changed to an aspartic acid, whereas in the study by Chang et al. (16), the alanine mutation was sufficient to show an effect. It is possible that the T cell assay is more sensitive than the tetramer-binding assay, however, in the hybridoma-expressing mutant K103A CD8 in the Chang study, the CD8 level was lower than that for wild type which may have contributed to the lower IL-2 production.
To determine whether human CD8 bound to MHC class I in a similar manner, we performed mutagenesis of the human CD8 residues in which the analogous mouse residues had shown an effect on binding to MHC class I. In general, the contacts with human CD8 and MHC class I appear to be similar, as with the CDR1, 2, and 3 loops involved in binding to MHC class I, although as of yet we have not identified mutants that showed the same dramatic increase in MHC class I binding. One possible explanation for this difference is that, unlike mouse CD8, human CD8 can be expressed on the cell surface as a homodimer that does not bind to MHC class I (17). To study the effect of mutations in mouse CD8, we ensured that the majority of CD8 expressed on the cell surface was in the form of CD8 heterodimers by transfecting twice as much CD8 as CD8. However, we could not do this for human CD8 because of the formation of CD8 homodimers, therefore, it is possible that the mixture of dimers on the cell surface prevented the detection of significant enhancement of tetramer binding to CD8. We are creating chimeric molecules to specifically express heterodimers to address this question.
Our results suggest that there are differences between CD8 and CD8 binding to MHC class I. We found that some mutations, e.g., R8A, have less effect when expressed as a CD8 heterodimer. A study by Wong et al. (25) showed that a chimeric protein of the Ig domain with the R8A mutation and the stalk of CD8 was sufficient to completely rescue the effect of this mutant in a hybridoma system. Therefore, it is possible that the shorter stalk of CD8 causes a change in the topology of CD8 binding to MHC class I. A point to be noted is that, in contrast to the observation by Wong et al. (25) where in they observed a complete rescue in T cell activation with the CD8 stalk and the R8A mutation, we did not see a complete rescue in MHC class I binding; these differences are likely due to the different assays used. It is also formally possible that CD8 can bind to MHC class I in two orientations, with the membrane proximal location being the preferred orientation, because in our studies with mutant CD8, wild-type CD8 show a partial, but not a complete, rescue. In mapping the binding site for an anti-CD8 Ab that enhances tetramer binding, we argued that the Ab stabilizes a conformation with a higher affinity for MHC class I (26). There may be alternative orientations/conformations that are selected for in vivo depending on changes in glycosylation during T cell activation or interaction with components of the TCR that select for a preferred orientation.
The addition of CD8-enhancing mutants to an Ag-specific CD8 T cells could provide a novel approach to improve the immune response against weak Ags such as those presented on tumor cells. CD8+ T cells are capable of recognizing tumor Ags and mediating an antitumor response. However, tumor-reactive T cells often have <10% of the activity that is observed against nonself viral Ags, in part due to the low immunogenicity of the tumor Ags. One goal of T cell immunotherapy is to increase the reactivity of T cells to these weak, or poorly presented, Ags. A number of different approaches have been used to try and manipulate the immune response to enhance tumor recognition and killing. These include, vaccination with known tumor Ags to increase the frequency of T cells that can react with tumor Ags, or in vitro manipulation of T cell specificity by modification of the TCR and chains to increase the affinity for Ag or manipulation of costimulatory signals (reviewed Refs. 27 and 28). There are advantages of modification of CD8 to enhance activity or lower the threshold for activation of T cells compared with altering TCR proteins. The modified CD8 could be added to any CD8 T cell line, unlike manipulation of the TCR and chains. Importantly, our initial studies suggest that the increase in affinity of CD8 for MHC does not override the requirement of Ag specificity. Therefore, we propose that addition of CD8 with mutations that enhance coreceptor function to T cells presents a novel immunotherapeutic approach for the treatment of cancer and chronic viral infections.
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Acknowledgments |
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Abs; Dr. D Kranz for the 2C TCR hybridoma; and Dr. H. Cheroutre for RMA-Kb. |
Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grant R01 CA048115 (to P.B.K.) and the Trudeau Fellowship at the Yale School of Medicine (to D.T.). 
2 Address correspondence and reprint requests to Dr. Paula B. Kavathas, Section of Immunology, Yale University School of Medicine, Anlyan Center S641, 300 Cedar Street, P.O. Box 208011, New Haven, CT 06520-8011. E-mail address: Paula.Kavathas{at}yale.edu
3 Abbreviations used in this paper: pMHC I, peptide-MHC class I; MFI, mean fluorescence intensity; BI, binding index.
Received for publication April 12, 2006. Accepted for publication June 29, 2006.
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References |
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versus CD8 expression. J. Immunol. 167: 2577-2584. and the nonclassical MHC class I molecule, TL. Science 294: 1936-1939. endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56lck complexes. J. Exp. Med. 194: 1485-1495. establishes a functional link between the T cell receptor and CD8. J. Biol. Chem. 278: 3257-3264. heterodimer and comparison with the CD8 homodimer. Immunity 23: 661-671. [Medline], but not mouse CD8, can be expressed in the absence of CD8 as a homodimer. J. Immunol. 164: 833-838. and HLA-A2. Nature 387: 630-634. [Medline] ectodomain fragment in complex with H-2Kb. Immunity 9: 519-530. [Medline] interact differently with 2-microglobulin of the class I molecules H-2Kb and thymic leukemia antigen, while similarly with their 3 domains. J. Immunol. 168: 3881-3886. -chain enhances the coreceptor function of CD8. J. Immunol. 171: 867-874. antibody 53.6.7 which enhances CD8-MHC class I interaction indicates antibody stabilization of a higher affinity CD8 conformation. Immunol. Lett. 93: 123-130. [Medline]This article has been cited by other articles:
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  D. Thakral, J. Dobbins, L. Devine, and P. B. Kavathas Differential Expression of the Human CD8{beta} Splice Variants and Regulation of the M-2 Isoform by Ubiquitination J. Immunol., June 1, 2008; 180(11): 7431 - 7442. [Abstract] [Full Text] [PDF]
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J. Huang, L. J. Edwards, B. D. Evavold, and C. Zhu Kinetics of MHC-CD8 Interaction at the T Cell Membrane J. Immunol., December 1, 2007; 179(11): 7653 - 7662. [Abstract] [Full Text] [PDF]
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) or a heterodimer (
) with wild-type CD8
