Role of a3 domain of class I MHC molecules in the activation of high- and low-avidity CD8 1 CTLs

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1 International Immunology, Vol. 19, No. 12, pp doi: /intimm/dxm111 Published by Oxford University Press Role of a3 domain of class I MHC molecules in the activation of high- and low-avidity CD8 1 CTLs Igor M. Belyakov 1, Steven Kozlowski 2, Michael Mage 3, Jeffrey D. Ahlers 1, Lisa F. Boyd 3, David H. Margulies 3 and Jay A. Berzofsky 1 1 Molecular Immunogenetics and Vaccine Research Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 2 Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA 3 Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA Keywords: cytotoxic T lymphocytes, MHC class I, avidity, epitope, peptide, TCR Abstract CD8 can serve as a co-receptor or accessory molecule on the surface of CTL. As a co-receptor, CD8 can bind to the a3 domain of the same MHC class I molecules as the TCR to facilitate TCR signaling. To evaluate the role of the MHC class I molecule a3 domain in the activation of CD8 1 CTL, we have produced a soluble 227 mutant of H-2D d, with a point mutation in the a3 domain (Glu227/Lys). 227 mutant class I peptide complexes were not able to effectively activate H-2D d -restricted CD8 T cells in vitro, as measured by IFN-g production by an epitope-specific CD8 1 CTL line. However, the 227 mutant class I peptide complexes in the presence of another MHC class I molecule (H-2K b ) (that cannot present the peptide) with a normal a3 domain can induce the activation of CD8 1 CTL. Therefore, in order to activate CD8 1 CTL, the a3 domain of MHC class I does not have to be located on the same molecule with the a1 and a2 domains of MHC class I. A low-avidity CD8 1 CTL line was significantly less sensitive to stimulation by the 227 mutant class I peptide complexes in the presence of the H-2K b molecule. Thus, low-avidity CTL may not be able to take advantage of the interaction between CD8 and the a3 domain of non-presenting class I MHC molecules, perhaps because of a shorter dwell time for the TCR MHC interaction. Introduction CD8 CTLs are a major effector for protection against many viral pathogens and tumors (1 10). Activation of CD8 + CTL is controlled by multiple receptor ligand interactions between antigen-presenting cells (APCs) and CD8 + T cells (11 18). Two signals are required for T-cell activation: signal 1 results from antigen-specific interaction between MHC peptide complexes on the APC with the TCR on the T-cell surface (19); signal 2 is mediated by interactions of costimulatory molecules on the APC with their receptors on CD8 + T cells. Activation of CD8 + CTL is strengthened by binding of the CD8 co-receptor with the a3 domain of MHC class I. In the presence of a weak signal 1 (because of low-affinity peptide) or sub-optimal signal 2, the activation of CD8 + T cells depends on the CD8 co-receptor; whereas in the presence of a strong signal 1 and signal 2, the co-receptor role of CD8 is redundant (20). CD8 promotes the formation of TCR MHC class I peptide complexes (21) and directly associates with Lck (22, 23). CD8 acts by augmenting signaling by focusing Lck into close proximity with the TCR (20, 22). In a study by Shen et al. (24), co-receptor and accessory functions on the surface of CTL were evaluated. It was demonstrated that the a3 domain-mutant class I peptide complexes were bound by CTL and triggered degranulation, however, to much lower levels than wild-type class I peptide complexes. While co-receptor functions of CD8 were diminished by a point mutation in the a3 domain of the MHC class I (Glu227/Lys), the ability of CD8 + T cells to be activated by TCR stimulation to bind class I molecules with high avidity (accessory function of CD8) was preserved (24). These data were strengthened by Knall et al. (25), who confirmed that CD8 functions much more efficiently as a coreceptor than as an accessory molecule for T-cell effector function. It was demonstrated that cells expressing a mutant H-2K b molecule required the addition of 100-fold more exogenous peptide than did cells expressing the wild-type molecule in order to elicit significant lysis (26). Fluorescence Correspondence to: I. M. Belyakov; igorbelyakov@yahoo.com Received 14 June 2007, accepted 4 October 2007 Transmitting editor: I. Pecht Advance Access publication 1 November 2007

2 1414 a3 Domain of MHC I for CD8 CTL activation resonance energy transfer (FRET) microscopy demonstrated transient interaction between CD3f and CD8b at the synapse between a T cell and an APC loaded with agonist peptide (18). Also non-stimulatory endogenous or exogenous peptides, presented simultaneously with specific peptides, enhanced the CD8 TCR interaction (18). However, the role of a3 domain of class I MHC molecules in the activation of high- and low-avidity CD8 + CTLs was never investigated before. Some previous work in the field had supported the conclusion that MHC molecules not presenting the peptide could interact with CD8 and contribute to the T-cell response (18,27 31), whereas other studies had come to the opposite conclusion (32 35). Therefore, the current study was undertaken in part to resolve the issue. In a study by Takeshita et al. (31), a cell-free antigenpresenting system was developed to quantitatively analyze the molecular interactions involved in CD8 + CTL recognition. Soluble MHC class I molecules (H-2D d ) on plastic were used to present an HIV peptide to an antigen-specific CD8 + CTL clone and induced production of IFN-c as a result of T-cell activation. It was demonstrated that the magnitude of T-cell activation showed first-order dependence on the concentration of the P18-I10 peptide but second-order dependence on that of the soluble H-2D d molecule. This study suggested that one MHC molecule can play more than one role in activating the CD8 + CTL since T-cell activation was more sensitive to the concentration of the MHC molecule than to that of peptide (31). Also it was demonstrated that an irrelevant MHC class I molecule (H-2K b ) augmented CD8 + T-cell responses at lower peptide concentrations (31) and changed the dependency of the response on MHC concentration from second order to first order, perhaps by reducing the dependence on the relevant MHC molecule to its role in presenting peptide. The co-receptor role of CD8 (CD8 a3 interaction) for the activation of high- versus low-avidity CTL is not well understood (36, 37). The role of the wild-type a3 domain of another MHC class I molecule (H-2K b ) (that cannot present the peptide) for activation of antigen-specific CD8 + CTL depends on the system studied. To investigate the role of the interaction of the a3 domain of MHC class I molecules with CD8 in the activation of the CD8+ CTL, we compared the activation of HIV-1 P18-I10-specific CD8 + CTL by soluble wild type MHC class I molecules (H-2D d ) and soluble H-2D d molecules with a mutation in position 227 (Glu to Lys) (227 mutant) that prevents binding of CD8 to the a3 domain. The soluble 227 mutant on plastic was not effective in activating the CD8 + T-cell line in vitro. However, the a3 domain from another MHC class I molecule (H-2K b ) could contribute to the activation of CD8 + CTL. Thus, for activation of CD8 + CTL, the a3 domain does not have to be located in the same molecule with the a1 and a2 domains of MHC class I presenting the peptide. A low-avidity CTL line was less well activated by the 227 mutant in the presence of H-2K b compared with a high-avidity CD8 + CTL line. This difference may be explained by more stable binding of a1 and a2 domains of the MHC class I peptide complex by the TCR of a high-avidity CD8 + CTL compared with a lowaffinity TCR MHC class I peptide interaction in low-avidity CTL. Methods Cell lines and cell purification The HIV-envelope gp160-specific CD8 + line was derived from BALB/c mice immunized with a recombinant vaccinia virus expressing HIV-1 gp160iiib (vpe16) (38), a kind gift of P. Earl and B. Moss. The spleens were aseptically removed and single-cell suspensions prepared by gently teasing them through sterile screens. The erythrocytes were lysed in Tris-buffered ammonium chloride and the remaining cells washed extensively in RPMI-1640 containing 2% fetal bovine serum (39, 40). Antigen-specific T cells were isolated from the spleen by multiple in vitro re-stimulations with irradiated syngeneic spleen cells pulsed with lm (for highavidity CD8 + CTL) or with 10 lm (for low-avidity CD8 + CTL) concentration of P18-I10 peptide (41, 42). Soluble class I MHC proteins and 227 mutant L-cell transfectants expressing either the H-2D d molecule (consisting of the a1, a2 and a3 domains of H-2D d and the C-terminal residues of the obligately soluble Q10b molecules) or the Glu 227-Lys mutant were cultured and supernatants harvested. H-2D d was purified as previously described (43, 44). Soluble class I MHC-presenting system The soluble class I MHC-presenting system was originally described by Takeshita et al. (31). Soluble H-2D d or 227 mutant of H-2D d was coated onto the 96-well microtiter plastic plate (Immulon-4, Dynatech, Chantilly, VA, USA), titrating from 0 to 3 lg per well in 50 ll of PBS for 2 h at 37 C. The plates were washed twice with PBS and blocked with 1% BSA (Sigma, St Louis, MO, USA) for 45 min. P18 peptide in 50 ll of 10% FCS complete T cell medium was added and the plates were incubated overnight at 37 C and 6% CO 2. The plates were washed with PBS three or four times. CTL lines (10 4 ) were added to each well in 10% FCS complete T cell medium. The supernatants were harvested after 24 h incubation at 37 C (31). ELISA for IFN-c The concentration of IFN-c in the supernatants was studied by an ELISA assay as previously described (45). CTL assay Cytolytic activity of CTL lines was measured by a 4-h assay with 51 Cr-labeled targets as described previously (46). P815 cells were used as targets cells. CD8 + CTL lines were assayed for lysis of target cells pulsed with different concentrations of antigen at an effector-to-target ratio of 10:1. For testing the peptide specificity of CTL, 51 Cr-labeled P815 targets were pulsed for 2 h with peptide at the beginning of the assay. The percent-specific 51 Cr release was calculated as (experimental release spontaneous release)/ (maximum release spontaneous release). Maximum release was determined from supernatants of cells that were lysed by addition of 5% Triton X-100. Spontaneous release was determined from target cells incubated without added effector cells (47).

3 Results Purified recombinant soluble class I molecules (H-2D d ) coated on plastic plates effectively presented peptide P18-I10 to the CD8 + CTL line specific for this peptide, as measured by IFN-c production by the line (Fig. 1). In this experiment, a high-avidity CD8 + CTL line was used. This CD8 + CTL line was raised from spleen cells of BALB/c mice, which were immunized with HIV-recombinant vaccinia virus (vpe16), by multiple in vitro re-stimulations with irradiated syngeneic spleen cells pulsed with lm P18-I10 peptide. The level of activation of CD8 + CTL was dependent on the P18-I10 peptide concentration, which was loaded onto the soluble class I molecules (Fig. 1). We noted that for our CD8 + CTL line, the optimal pulsing concentration of P18-I10 peptide was 1 lm, and we used this concentration of P18- I10 peptide in our future experiments (Fig. 1). When the lower concentrations of P18-I10 peptide were used ( lm), the production of IFN-c was slightly reduced (Fig. 1). As a control, we studied the soluble H-2K b molecules and noted that H-2K b was not able to present P18-I10 peptide to CD8 + CTL line (data not shown). The activation of CD8 + CTL in vitro was greatly dependent on the concentration of soluble H-2D d molecules (wild type) on plastic. Presentation of P18-I10 peptide to the CD8 + CTL line was detectable (by measurement of IFN-c concentration in cell a3 Domain of MHC I for CD8 CTL activation 1415 culture media) when as little as 0.1 lg per well of wild type soluble H-2D d was used. A 0.2 lg per well raised the IFN-c level production by CD8 + CTL line to plateau level (Fig. 2A). To investigate the role of the a3 domain of MHC class I molecules in the activation of CD8 + CTL, we used soluble H-2D d molecules with a mutation in position 227 (Glu to Lys) (227 mutant) that prevents binding of CD8. Activation of a highavidity CD8 + T-cell line by the 227 mutant class I peptide Fig. 1. Activation of a high-avidity CTL lines on MHC-coated plastic depends on P18-I10 peptide and MHC concentration. A high-avidity CD8 CTL line from spleen cells of BALB/c mice immunized with HIVrecombinant vaccinia virus (vpe16), grown by multiple in vitro restimulations with irradiated syngeneic spleen cells pulsed with lm P18-I10 peptide, was activated on plastic with different concentrations of P18-I10 peptide (0.01, 0.1, 1 and 10 lm), loaded on soluble class I molecules coated on the plastic (0, 0.01, 0.15 and 0.18 lg per well of H-2D d ). P18-I10 peptide in 50 ll of 10% FCS complete T cell medium was added and the plates were incubated overnight at 37 C and 6% CO 2. The plates were washed with PBS three or four times. CTL lines (10 4 ) were added to each well in 10% FCS complete T cell medium. Presentation of P18-I10 peptide to CD8 + CTL line was detectable by measurement of IFN-c concentration in cell culture media after 24 h incubation at 37 C (31). These experiments were performed twice with comparable results. Fig. 2. The a3 domain of MHC class I molecules does not have to be located in the same molecules with the a1 and a2 domains of MHC class I presenting the peptide in order to activate CD8 + CTL. Another type of MHC class I molecule can provide the a3 domain for the activation of CD8 + CTL. The a3 domain from H-2K b molecules compensates for the altered signal from the dysfunctional a3 domain of mutant H-2D d. (A) 227 mutant dose-dependent activation of CD8 + CTL, while the concentration of H-2K b is constant (0.5 lg per well). (B) a3 domain of K b dose-dependent activation of CD8 + CTL, while the concentration of 227 mutant is constant (1.2 lg per well). These experiments were performed twice with comparable results.

4 1416 a3 Domain of MHC I for CD8 CTL activation complexes was diminished (Fig. 2A). 227-mutant class I peptide complexes were not able to effectively activate CD8 T cells in vitro, asmeasuredbyifn-c production by the P18- I10-specific CD8 + CTL line. The response of the CD8 + CTL reached a plateau level at a concentration of the 227 mutant class I peptide complexes of 2.5 lg per well (Fig. 2A). However, the magnitude of the CD8 + CTL activation at the peak by 227 mutant class I peptide complexes was about three times lower compared with activation of CD8 CTL by the wild-type H-2D d (Fig. 2A). We asked whether the a3 domain from other MHC I molecules different from H-2D d would compensate by a nonspecific signal from the other a3 domain to CD8 molecules. It is known that H-2K b molecules cannot present the P18-I10 peptide to CD8 + CTL (48; Fig. 2B). We directly added the standard concentration of soluble H-2K b class I molecules (0.5 lg per well) to wells which were coated with the 227 mutant of H-2D d. In this experiment, we titrated the concentration of 227 mutant from 0 to 3 lg per well (Fig. 2A). As a control, we compared the activation of CD8 + CTL cells by wild-type H-2D d molecules that were loaded with P18-I10 peptide. A concentration of 0.2 lg per well of wild type of soluble H-2D d in presence of I10 peptide elevated the level of IFN-c to a plateau. The range of concentrations of 227 mutant H-2D d from 0 to 1.2 lg per well in the presence of 0.5 lg per well of soluble H-2K b molecules was not sufficient to significantly activate CD8 + CTL cells in vitro (Fig. 2A). When we used concentrations of the 227 mutant higher than 1.5 lg per well in the presence of 0.5 lg per well of H-2K b, a significant up-regulation of IFN-c production by CD8 + CTL was observed. The level of activation of CD8 + CTL was almost on a plateau level when 2 lg per well (and higher) of 227 H-2D d mutant class I peptide complexes in presence of H-2K b molecules were used and was not significantly different from the response to wild-type H-2D d loaded with peptide (Fig. 2A). Thus, the co-receptor signal for CD8 + CTL activation on plastic by mutant H-2D d can be compensated by the presence of the a3 domain from another MHC class I molecule. Also we performed the analysis with a standard suboptimal concentration of 227 mutant (1.2 lg per well) on the plastic together with a titrated range of H-2K b MHC class I molecules (from 0 to 3 lg per well) in order to activate the CD8 + CTL in vitro (Fig. 2B). A concentration of H-2K b of 0.5 lg and higher in the presence of the 227 mutant class I peptide complexes can induce the activation of CD8 + CTL (Fig. 2B). In contrast, H-2K b alone does not stimulate at any concentration. Thus, the non-specific signal provided by the a3 domain of MHC class I molecules (through the interaction with CD8 molecules on CTL) in case of the a3 deficiency can be fully compensated by the a3 domain from other MHC class I molecules (such as H-2K b ). In order to activate CD8 + CTL, the a3 domain of the MHC class I molecule does not have to be located on the same molecules with the a1 and a2 domains. Other types of MHC class I can provide the a3 domain to work with the 227 mutant. We developed high- and low-avidity CD8 + CTL lines to compare their sensitivity with the a3 domain on a different MHC molecule. These CD8 + CTL lines were raised from spleen cells of BALB/c mice immunized with HIV-recombinant vaccinia virus (vpe16) by multiple in vitro re-stimulations with irradiated syngeneic spleen cells pulsed with lm (for high-avidity CD8 + CTL) or with 10 lm (for low-avidity CD8 + CTL) concentration of P18-I10 peptide and assayed for lysis of target cells pulsed with different concentrations of antigen at an effector-to-target ratio of 10:1 (Fig. 3A). The high-avidity CTL line was raised on APCs pulsed with the lowest concentrations of P18-I10, whereas the CTL lines raised on APCs pulsed with the highest concentration of P18-I10 required much higher concentrations of peptide on the targets to achieve the same level of lysis and are thus lower avidity CD8 + CTL (Fig. 3A). Also in our experiments, we used high- and low-avidity CTL lines with a similar level of CD8 expression to exclude that parameter as a variable. We characterized the activation of low-avidity CD8 + CTL under the same conditions as high-avidity CD8 + CTL in the presence of titrated amounts of the 227 mutant and constant H-2K b (0.5 lg per well) (Fig. 3B). The low-avidity CTL was not activated even in the presence of a high concentration of 227 mutant class I peptide complexes in the presence of H-2K b molecules. One of the predictions was that low-avidity CD8 + CTL required more time for antigen recognition and activation. However, this was not the case. We studied CD8 + T-cell activation at different time points and found that low-avidity CD8 CTL cannot be activated even after a long in vitro stimulation on plastic with the 227 mutant MHC class I molecules loaded with P18-I10 peptide (Fig. 3C). These data demonstrate that the avidity of the CD8 + CTL is a most important factor for effective CD8 + T-cell activation in vitro and in vivo. Discussion Activation of CD8 T cells required at least two signals. Signal 1 results from antigen-specific recognition of the MHC class I peptide complex on the APC by the TCR, whereas signal 2 is provided by the non-specific interaction of co-stimulatory molecules on APC with their receptors on CD8 CTL (16, 20). Signal 1 alone is not sufficient to activate fully naive CD8 T cells despite the induction of TCR down-regulation. Activation and differentiation of CD8 + T cells at low MHC peptide densities required signal 2 with both B7 and Intercellular adhesion molecule-1 on the same APC (20). CD8 can bind to the same MHC class I molecules as the TCR to improve the efficiency of TCR signaling and thus serve as a co-receptor. Also CD8 CTL can be activated by TCR stimulation to bind to MHC class I molecules with high avidity, including CD8 interaction with MHC class I molecules not recognized by the TCR as antigenic complexes, to enhance CD8 + CTL responses. In this context, CD8 can serve as an accessory molecule. The CD8 co-receptor interacts with MHC class I molecules through an acidic loop in the a3 domain and this interaction is required for effective CD8 + CTL activation (35, 49, 50) and in positive and negative selection of developing T cells (51). In a study by Shepherd et al. (52), H-2K b -specific recognition of H-2K b structural mutant (a single Glu/Lys amino acid substitution at position 222 in the H-2K b a3 domain) by CD8-independent CTL was unaltered, while the response by CD8-dependent CTL was completely abrogated. However, the mechanism of CD8 co-receptor ligand interactions is not very well understood.

5 a3 Domain of MHC I for CD8 CTL activation 1417 Since endogenous class I molecules were expressed by all the transfected cell lines, the study by Connolly et al. (34) concluded that CD8 and the abtcr must interact with the same class I molecule. For recognition by CD8-dependent CTLs, residue 227 must be either glutamic acid or aspartic acid and cannot be either basic or uncharged (34). Others suggested that the CD8 and TCR must co-localize in lipid rafts (17). Our study demonstrates that a CD8 + CTL line can significantly benefit from the a3 domain on a non-tcr-binding MHC. A likely mechanism for this is an interaction with CD8 co-localized with TCR in lipid rafts (Fig. 4). Probably, it is still not as effective as true co-localization on the same MHC because Lck is moved closer to its target in the lipid rafts. However, this effect of CD8 co-localization with TCR in lipid rafts may be significantly amplified by the interaction of co-stimulatory molecules on APC with their receptors on CD8 CTL. Also it is possible that an isolated T-cell clone or restricted population may have a low, but otherwise undetectable a1/a2 domain interaction with H-2K b and CD8 activation might be mediated by binding to a second TCR that is close to the H-2D d -binding TCR. However, as it was discussed above, our previous studies have never detected any hint of recognition of H-2K b by high- and/or low-avidity P18-I10 CTL lines or by primary T cells from immunized animals and it would seem exceedingly unlikely for the whole population to behave this way. The quantitative study of peptide MHC class I TCR interaction is difficult because it is not always possible to titrate the concentration of MHC class I molecules. This is why the development of a cell-free antigen-presenting system was needed. In the study by Takeshita et al. (31), the titration of MHC class I molecules was done by changing the density of recombinant soluble H-2D d molecules coated on the plastic plate, and we exploited this system in our study. Peptide and MHC molecules together were sufficient for CD8 + CTL activation in vitro, without the presence of any co-stimulatory molecules. Also MHC class I peptide interaction was accomplished in the absence of serum, and free peptide was washed away before T cells were added (31). The peptide binding to class I MHC required h to reach halfmaximal responses, and once the peptide was bound to MHC molecules, the complexes were very stable at least for 24 h (31). This study demonstrated that the T-cell response was more sensitive to varying the H-2D d concentration than to varying the peptide concentration. These results suggested that the H-2D d molecule played more than one role in the Fig. 3. Generation of low- and high-avidity CD8 + CTL lines in vitro. (A) CD8 + CTL lines were raised from spleen cells of BALB/c mice immunized with HIV-recombinant vaccinia virus (vpe16) by multiple in vitro re-stimulations with irradiated syngeneic spleen cells pulsed with lm (for high-avidity CD8 + CTL) or with 10 lm (for low-avidity CD8 + CTL) concentration of P18-I10 peptide and assayed for lysis of target cells pulsed with different concentrations of antigen at an effector-to-target ratio of 10:1. (B) The low avidity of P18-I10-specific CD8 + CTL line was activated less effectively by the titrated amount of 227 mutant class I peptide complexes plus H-2K b (concentration of H-2K b is constant, 0.5 lg per well) compared with the high-avidity P18-I10-specific CD8 + CT line. (C) Kinetics of activation of highversus low-avidity CD8 + CTL lines by 227 mutant H-2D d plus P18-I10 peptide. Diamonds, high-avidity CTL; squares, low-avidity CTL. These experiments were performed twice with comparable results.

6 1418 a3 Domain of MHC I for CD8 CTL activation Fig. 4. Schema of activation of CD8 + CTL by peptide MHC class I complexes on APC. The co-receptor signal for CD8 + CTL activation by mutant H-2D d can be compensated by the presence of the a3 domain from another MHC class I molecule, co-localized in the lipid raft and drawing CD8 into the synapse. interaction (31). The interaction between the CD8 molecule and a non-polymorphic region of the H-2D d molecule (a3 domain) was suggested as the second role for the H-2D d in CD8 + T-cell activation in addition to presenting peptide to the TCR (31). This was concluded based on the effect of another class I MHC molecule, H-2K b, that cannot present the peptide, on the shape of the titration curve, changing it from second order to first order in the H-2D d concentration. Also, a recent FRET microscopy study by Yachi et al. (18) demonstrated non-antigen-specific recruitment of CD8 into the synapse between a T cell and an APC, as reported earlier for CD4 T cells (53). The non-stimulatory peptides induced CD8 clustering to the synapse as efficiently as the antigenic peptide in the conjugates (18). However, the role of non-specific recruitment of CD8 for activation of high- and low-avidity CD8 + CTL was never demonstrated before. CD8 + CTL that can recognize peptide MHC only at high antigen concentration are defined as low-avidity CD8 + CTL, while those that can be activated by low concentration of antigen are termed high-avidity CD8 + CTL (36, 41, 54, 55). CD8 + CTL functional avidity has been shown to be an important determinant of in vivo protective efficacy. It was demonstrated that high-avidity CD8 + CTLs are essential for effective clearance of viral infection (41, 42,56 61) and elimination of tumor (62 66). It was also shown that a high-avidity CD8 + CTL expressed a higher level of CD8b + compared with a low-avidity CD8 + CTL (67). However, the role of CD8 molecules and CD8 a3 interaction in activation of high- versus low-avidity CTL is not very well understood (36,68 71). To further explore this role in current study, we characterized the direct role of CD8 a3 interaction (without involvement of co-stimulatory molecules) for activation of high versus low avidity of CD8 + CTL, using a mutant H-2D d with residue Glu 227 replaced with Lys, preventing the interaction with CD8. The soluble 227 mutant H-2D d alone on plastic was not able to effectively activate either the high- or low-avidity CD8 + T-cell line in vitro. However, a non-specific signal provided by the a3 domain of other MHC class I molecules (H-2K b ) not presenting peptide can restore the activation of high-avidity CD8 + CTL. Thus, for such activation of CD8 + CTL, the a3 domain does not have to be located on the same molecule with the a1 and a2 domains of MHC class I molecule to activate high-avidity CTL (Fig. 4). In contrast, the low-avidity CTL line was less well activated by the H-2D d 227 mutant plus H-2K b. This suggests that the lowavidity CTL much more depend on co-receptor CD8 a3 interaction with the same MHC molecule and probably need the presence of some additional co-stimulatory factors/molecules which are absent in our system. Also, during the activation process on the plastic with soluble MHC class I molecules, cells may not be able to form as many synapses as observed in situ during T-cell activation by APC. Further, high-affinity binding by a1 and a2 domains of the MHC class I peptide complex to the TCR is more stable compared with low-affinity interaction, which may dissociate more rapidly. Thus, the longer dwell time of the TCR CD8 + CTL of high avidity may allow time for the non-presenting MHC molecule to bring CD8 molecules into the lipid raft with their associated kinase Lck to permit phosphorylation of f chains of the TCR of the high-avidity CD8 CTL, whereas the dwell time may be too short for the low-avidity CTL. Thus, our study demonstrated that in order to activate CD8 + CTL, the a3 domain of MHC class I does not have to be located on the same molecule with the a1 and a2 domains of MHC class I. The 227 mutant class I peptide complexes in the presence of the H-2K b molecule significantly more effectively activated high-avidity CD8 + CTL. Funding Intramural program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health. Acknowledgements We thank Patricia Earl and Bernard Moss (NIAID, Bethesda, MD, USA) for the gift of vaccinia virus vpe16. We thank Kannan Natarajan for critical reading of the manuscript and helpful suggestions.

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