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Peptide Requirement for CTL Activation Reflects the Sensitivity to CD3 Engagement: Correlation with CD8 Versus CD8 Expression¹

Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In our previous studies, CTL that were sensitive to low concentrations of peptide Ag were found to be far superior to those requiring high concentrations of Ag for reducing viral burden when adoptively transferred into SCID mice. Thus it is important that we understand the mechanisms that control the requirement for peptide Ag with the long-term goal of selectively expanding these exquisitely sensitive cells in vivo. Although TCR affinity is one parameter that can affect the CTL sensitivity for Ag, we investigated whether additional mechanisms may also be involved. In studies using a TCR transgenic mouse model, we successfully generated CTL with identical TCR affinity that possess distinctly different activation requirements. Using both peptide Ag and anti-CD3 Ab to activate the CTL lines of high vs low avidity, we found that the variations in activation threshold are the result of differences in the required number of engaged TCR. Additionally, we have observed that the ratio of CD8 to CD8 is significantly greater in CTL lines that are more sensitive to TCR engagement, which may contribute to the lower activation threshold of these CTL following CD3 engagement. These studies identify a novel mechanism by which the activation requirements of Ag-specific CTL are determined by demonstrating a direct correlation between the sensitivity to TCR engagement, the expression of levels CD8 vs , and the amount of peptide Ag required to reach the threshold for activation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The sensitivity to peptide Ag is a critical parameter in determining the efficacy of viral clearance by CTL. Our previous data in which viral clearance was assessed in a SCID mouse model demonstrated that although CTL that could be activated in response to low concentrations of Ag were capable of reducing viral burden by 3 logs in 3 days, CTL that required high concentrations of Ag were unable to effect viral clearance (1). This correlation between the sensitivity to peptide Ag and the efficiency of viral clearance has been subsequently confirmed in studies of lymphocytic choriomeningitis virus (LCMV)³ infection (2).

In our previous studies, cells that were responsive to low concentrations of peptide Ag were termed high-avidity CTL, whereas those cells that required significantly more Ag to become activated were referred to as low-avidity CTL. The assignment of avidity as it relates to Ag dose is determined functionally by the amount of peptide required for activation or effector function (1, 3, 4, 5, 6, 7, 8) and is a measure of the overall strength of the interaction between a CTL and a target cell. Thus any molecule on the surface of a CTL has the potential to contribute to the overall functional avidity of that cell. The avidity of CTL that are generated in vitro is determined by the amount of peptide used for stimulation (1). Stimulation with APC presenting a high density of peptide/MHC complexes will select for CTL of low avidity, whereas stimulation with APC displaying a low density of peptide/MHC complexes will selectively activate CTL of high avidity. When established, CTL avidity cannot be modulated, i.e., low-avidity CTL cannot become high avidity by altering the stimulation conditions. Thus, avidity appears to be a fixed phenotype in a previously activated cell.

The full range of parameters that may influence the Ag requirements (functional avidity) for CTL activation is not known. Certainly TCR affinity has been shown to play a significant role in determining the sensitivity of a CTL to peptide Ag (9, 10). Analyses using tetramers of peptide/MHC complexes have found a correlation between tetramer binding, which is accepted as a measure of TCR affinity, and the amount of Ag required to elicit a functional response (9, 10). Specifically, a correlation with increased tetramer staining and increased sensitivity to antigenic peptide was observed in the analysis of recall responses using a peptide from Listeria monocytogenes (10). Comparison of polyclonal populations of cultured CTL showed that the dissociation rate of tetramer was slower in cells from secondary responses when compared with primary responses. This correlated with an increase in the peptide sensitivity of CTL in the recall response, thus suggesting that expression of a high-affinity TCR produces a high-avidity CTL. In a study by Yee et al. (9), PBMC were stimulated with a tumor-specific peptide and resulting cultures were sorted for tetramer high and tetramer low cells based on the ability of these cells to bind fluorescently labeled peptide/MHC tetramers. When tested functionally for the ability to lyse tumor target cells, the tetramer-high population was more efficient than the tetramer-low population. However, when CTL clones were generated from these populations, a number of the CTL generated from the tetramer-high populations were incapable of recognizing tumor cells efficiently. Furthermore, there were also some tetramer-low CTL that could efficiently lyse tumor targets. Thus, these data suggested that factors other than TCR affinity can be important in determining the sensitivity to peptide Ag.

In a separate study by Al-Ramadi et al. (11), the affinity of TCR for surface peptide/MHC molecules was altered by creating monosubstituted peptide variants. When the functional responses of CTL to APC pulsed with these peptide variants was measured, it was discovered that although there were some peptide/MHC complexes that bound TCR with higher affinity relative to the nonvariant peptide, the increased binding affinity did not correlate with an increased functional response. The lack of correlation between TCR-peptide/MHC affinity and effector function suggests that TCR affinity is not the only determinant that governs the activation of CTL.

In addition, in a previous report using anti-CD3 Ab to redirect lysis in a panel of CTL lines, we found that CTL requiring high or low concentrations of Ag to achieve effector function can differ in their requirement for CD3 engagement (1). The amount of anti-CD3 Ab required for lysis by lines generated on high concentrations of peptide was significantly greater than the amount of Ab required by lines capable of responding to much lower concentrations of Ag. These data implied that the sensitivity to TCR engagement or cross-linking parallels the peptide requirements for CTL activation. However, the TCR affinity of the CTL in these lines was unknown. Thus, the relative contribution of the sensitivity to TCR engagement and the TCR affinity to the functional avidity in these lines could not be determined.

In this report we have explored the hypothesis that CTL with distinct requirements for peptide Ag can be generated independently of TCR affinity. To address other mechanisms that may control the amount of Ag required for activation, we used a LCMV TCR transgenic (P14) crossed onto a recombinase-activating gene 2 (RAG2)^-/- background as a model. The TCR transgene was established on a RAG2^-/- background to prevent generation of CTL expressing a TCR derived from rearrangement of endogenous TCR genes. As CTL generated from these mice share a common TCR with a defined affinity, any differences in activation requirements must be the result of mechanisms other than differences in the binding of TCR to peptide/MHC. Using the method we established previously for the generation of CTL lines of various avidity from wild-type mice, we found that CTL that are highly sensitive to peptide Ag and relatively less sensitive to peptide could be generated in the presence of a common TCR. Because the TCR affinity is equivalent, we have chosen to refer to these lines as highly sensitive (HS) or less sensitive (LS) to peptide in lieu of using the terms high avidity or low avidity. We have shown that the amount of peptide required for activation in these lines is controlled by the sensitivity of the CTL to TCR cross-linking for all effector functions tested, including lysis, IFN- production, and TCR down-regulation. In addition to the differences in dose response for both peptide Ag and anti-CD3 Ab, we have identified a disparity in the relative expression of CD8 and CD8 between HS and LS Ag-specific CTL. Both HS and LS cells express equivalent levels of CD8. However, HS cells express significantly more CD8 than LS CTL. The increased expression of CD8 correlates with the enhanced sensitivity of HS cells to CD3 cross-linking. These findings expand our understanding of the Ag requirements of CTL and may provide new insights into the activation and expansion of CTL in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and cell lines

C57BL/6 mice were purchased from Frederick Cancer Research and Development Center (Frederick, MD). The TCR LCMV P14/rag-2 mice were obtained from Taconic Farms (Germantown, NY). P815 is a DBA/2-derived mastocytoma and EL4 is a C57BL/6-derived thymoma. The LCMV P14 peptide (KAVYNATM) encompasses residues 33–41 of the gp33 protein and was synthesized at the Comprehensive Cancer Center Protein Analysis Core Laboratory at Wake Forest University School of Medicine.

Generation of CTL lines

For lines generated from the transgenic mice, 2 x 10⁶ spleen cells were cocultured with 3.5 x 10⁶ C57BL/6 splenocytes (2000 rad irradiated) previously pulsed with either low (10^-5 M) or high (10^-10 M) concentrations of LCMV (P14) peptide. Cultures were maintained in 24-well plates containing 2 ml of RPMI 1640 medium supplemented with 2 mM L-glutamine, 0.1 mM sodium pyruvate, nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, 2-ME (0.05 mM), 10% FCS, and 10% T-stim (Collaborative Biomedical Products, Bedford, MA). CTL lines were established from primary cultures and were maintained by weekly restimulation of 3–5 x 10⁵ cells/well in the presence of 5 x 10⁶ irradiated (2000 rad) C57BL/6 spleen cells pulsed with the appropriate concentration of peptide.

⁵¹Cr-release assay

Target cells (1 x 10⁶) were labeled with 300 µCi of Na₂⁵¹CrO₄ in 200–250 µl for 2 h at 37°C. In some cases, targets were pulsed with graded concentrations of peptide during labeling. Cells were then washed three times and added at 3000 cells/well along with the appropriate number of effector cells in 96-well round-bottom plates. For redirected lysis assays P815 target cells were incubated with 300 µCi/1 x 10⁶ cells for 2 h at 37°C. Following incubation, cells were washed and plated at 3000 cells/well. Anti-CD3 Ab (clone 145-2C11) was added to wells at various concentrations and allowed to incubate for 15 min at 37°C. CTL were then added at an E:T ratio of 3:1 After 4 h, supernatants were harvested and counted in a gamma counter. The mean of triplicate samples was calculated, and percent ⁵¹Cr release was calculated according to the following equation: Percent specific ⁵¹Cr release = 100 x [(experimental ⁵¹Cr release - control ⁵¹Cr release)/(maximum ⁵¹Cr release - control ⁵¹Cr release)], where experimental ⁵¹Cr release represents counts from target cells mixed with effector cells, control ⁵¹Cr release represents target cells mixed with medium alone (spontaneous release), and maximum ⁵¹Cr release represents counts from target cells exposed to 2.5% Triton X-100.

IFN- ELISA

Six days following routine stimulation, CTL were plated at 5 x 10⁴/well in a 96-well round-bottom microtiter plate. Irradiated (2000 rad) C57BL/6 splenocytes previously pulsed with peptide and washed three times were added at 3 x 10⁵/well. For anti-CD3-mediated IFN- production, 5 x 10⁴ CTL were incubated in 96-well flat-bottom plates that had previously been incubated overnight at 4°C with various concentrations of anti-CD3 Ab (clone 2C11; BD PharMingen, San Diego, CA) followed by PBS washing. Incubation of CTL with peptide-pulsed splenocytes or anti-CD3 Ab occurred at 37°C for 24 h in a 5% CO₂ incubator. Supernatant (50 µl) was harvested at 24 h and assayed for the presence of IFN-. The OptEIA Ab set (BD PharMingen) was used according to the manufacturer’s directions. Concentrations of IFN- were calculated based on the standard curve run concurrently in the assay.

Flow cytometry

For flow cytometric analysis, 2 x 10⁵ cells were washed and resuspended in PBS containing 2% FCS. Cells were incubated on ice with the appropriate Ab for 30 min and washed. Where necessary, a secondary reagent was then added for an additional 30 min and the cells were again washed. Biotin-conjugated anti-V Abs, anti-CD3 (clone 2C11), avidin-PE, and anti-hamster FITC Abs were obtained from BD PharMingen. CD8 (clone CT-CD8a), CD8 (clone CT-CD8b), and LFA-1 (clone I21/7) Abs were obtained from Caltag Laboratories (Burlingame, CA). For tetramer binding studies, CTL were incubate with tetramer on ice for 30 min, washed, and directly analyzed by flow cytometry. Tetramers were a gift of Drs. J. Grayson and R. Ahmed (Emory University, Atlanta, GA). Samples were analyzed on a FACStar (BD Biosciences, Mountain View, CA).

TCR and CD8 down-regulation assay

Flat-bottom (96-well) plates were coated with various concentrations of 2C11 (anti-CD3) Ab in PBS overnight at 4°C. Plates were washed three times in PBS and blocked in 10% FCS for 1 h at room temperature. CTL were placed over a Ficoll gradient and added to wells at 2 x 10⁵/well. Cultures were incubated in a 5% CO₂ incubator at 37°C for 5 h. After incubation, CTL were transferred to 96-well round-bottom plates for staining. For peptide-specific down-regulation, EL4 cells that had been pulsed with graded concentrations of LCMV peptide for 2–3 h and washed three times in PBS were cocultured with CTL (2 x 10⁵/well) for 5 h at 37°C in a 5% CO₂ incubator. TCR surface expression was determined by staining with biotinylated anti-V8 followed by streptavidin-PE, and CD8 expression was measured by FITC-conjugated anti-CD8 or CD8 Ab. Samples were analyzed by flow cytometry on a FACStar flow cytometer.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CTL lines with different requirements for Ag-induced activation can be generated from TCR transgenic x RAG2^-/- mice

To investigate mechanisms other than TCR affinity that are capable of influencing the sensitivity of CTL to peptide, we used a model system in which the TCR affinity was constant and thus the TCR could not contribute to any functional differences based on the ability to bind peptide/MHC. We chose the TCR transgenic mouse expressing the TCR specific for the LCMV gp P14 peptide presented by H-2D^b. This mouse has been crossed onto a RAG2^-/- background to prevent any possibility of endogenous TCR rearrangement and expression. Splenocytes from these mice were stimulated in vitro with APC that had been pulsed with either high (10^-5 M) or low (10^-10 M) concentration of peptide. In nontransgenic mice this method has been shown to result in the selective expansion of LS or HS CTL lines, respectively (1). We hypothesized that if factors other than the TCR affinity were involved in the control of sensitivity to Ag, then we would be able to generate both HS and LS CTL lines using the TCR transgenic mice. Lines established by this method were first tested for their expression of TCR. In Fig. 1A, the expression levels of TCR were measured by flow cytometric analysis using a mAb specific for the V portion of the TCR transgene. Expression levels of TCR between HS and LS lines were strikingly similar. In addition to measuring TCR expression, the surface levels of the CD3 portion of the TCR complex were also determined. It was important to test for the expression of CD3 in addition to TCR , as a previous report demonstrated the association of multiple heterodimers with a single CD3 molecule (12). Differences in the stoichiometry of /CD3 in the TCR complex between high- and low-avidity CTL could result in differences in activation efficiency. Fig. 1B demonstrates equivalent expression of CD3 on the surface of HS and LS cells. To further characterize the TCR on HS and LS lines, tetramer analysis was performed to determine whether the lines were capable of binding peptide/MHC with similar efficiency. It was possible that differences in the higher order membrane arrangement of the transgenic TCR could allow for differences in tetramer binding even though the TCR affinity for peptide/MHC is identical. Fig. 1C shows similar tetramer binding between HS and LS lines, establishing that HS and LS lines bind peptide/MHC complexes equivalently.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. HS and LS lines express similar levels of TCR and bind tetramer equivalently. HS (dotted) and LS lines (solid) were analyzed on day 6 post stimulation. A, Surface expression of TCR was determined by staining with an anti-V8 Ab specific for the transgenic TCR. B, Expression of the CD3 portion of the TCR complex was determined using an anti-CD3 Ab (clone 145-2C11). C, HS and LS lines bind peptide/MHC complexes equivalently. P14 peptide/Db fluorescently labeled tetramers were incubated with HS and LS cells on ice, washed, and analyzed by flow cytometry.

View larger version (18K): [in this window] [in a new window]   FIGURE 2. HS and LS CTL sharing a common TCR require different concentrations of Ag to achieve effector function. A, EL4 cells were pulsed with a range of LCMV peptide concentrations and used as targets in a chromium release assay. CTL of HS (•) or LS () functional phenotypes expanded from TCR transgenic mice were used as effectors at an E:T ratio of 3:1. B, Splenocytes from nontransgenic mice were pulsed with varying concentrations of LCMV peptide and used as stimulators in an IFN- assay with CTL of HS or LS phenotype. Supernatants were harvested at 24 h, and IFN- production was measured by ELISA. CTL lines were assayed on day 6 post stimulation.

The above results suggested that the differences in peptide requirement were the result of differences in the number of TCR molecules that must be engaged to evoke effector function or activation. To test this possibility, the dose response to anti-CD3 Ab (2C11) was measured. CTL of HS and LS functional phenotypes were incubated in the presence of FcR⁺ P815 target cells and titrated concentrations of 2C11 Ab. This assay effectively bypasses TCR engagement of peptide/MHC and instead activates via direct engagement of the CD3 molecule. Fig. 3 shows the dose response to 2C11 (anti-CD3 Ab) for both lytic activity (Fig. 3A) and IFN- production (Fig. 3B). In the redirected lysis assay, LS cells required 6-fold more 2C11 than HS cells to reach half-maximal lysis. When IFN- production was measured as a function of 2C11 concentration, LS cells reached half-maximal production at a concentration of anti-CD3 Ab that was 10-fold higher than HS CTL. Again, the differences in the dose-response curves were not the result of differences in the expression of CD3, as the HS and LS CTL lines display similar expression levels of CD3 on their surface (Fig. 1C). Thus, at each concentration of anti-CD3 Ab tested in Fig. 3, the same number of CD3 molecules should be engaged. It is clear from these data that the number of TCR molecules that must be engaged to trigger a functional response from an individual line correlates with the HS or LS phenotype previously assigned based on the response to peptide Ag. These data, showing a difference in the dose-response curve to anti-CD3 Ab, support a model in which differences in the signaling capacity of the CTL in the individual lines is responsible for the observed differences in peptide requirement. The difference in the concentration of peptide (100–1000x) vs anti-CD3 Ab (6- to 10-fold) required between HS and LS lines are expected, as the amount of peptide loaded onto the D^b molecule following incubation with log dilutions of peptide is not linear, i.e., 10x more peptide does not equal 10x more peptide/MHC complexes at the cell surface (data not shown). Furthermore, the affinity of anti-CD3 Ab for the CD3 molecule is much greater than the TCR affinity for peptide/MHC and is a more efficient interaction. Additionally, activation via engagement of the TCR with peptide/MHC complexes differs from activation by cross-linking of the CD3 complex with Ab in that stimulation with peptide-pulsed APC allows for CD8 binding. Therefore, although we can conclude that differences in the sensitivity to TCR cross-linking contribute to the observed differences in the requirement for peptide Ag, it is also possible that other mechanisms contribute to the differential sensitivity of these lines to TCR engagement.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. HS functional phenotype correlates with greater sensitivity to TCR cross-linking. A, Dose-dependent cytotoxic activity of HS (•) or LS () CTL lines. Fc receptor-positive P815 cells were incubated with titrated amounts of anti-CD3 Ab and used as targets in a redirected lysis assay. CTL were added at an E:T ratio of 3:1. The percent maximal lysis was determined for each CTL line as a function of anti-CD3 Ab concentration. B, IFN- secretion by HS (•) or LS () CTL following culture with titrated concentrations of immobilized anti-CD3 Ab. Supernatants were collected at 24 h, and IFN- concentration was measured by ELISA. CTL were assayed on day 6 post stimulation.

Signal transduction by TCR molecules leads to TCR down-regulation (13). Thus TCR internalization can be used to deduce the number of triggered TCR involved in T cell activation (14). The HS and LS lines in the current study use an identical TCR; therefore, the efficiency with which the TCR engages peptide/MHC complexes should be equivalent. This has been confirmed by tetramer analysis in Fig. 1C. Thus, one might predict that the TCR internalization as a result of peptide stimulation would be the same between the lines.

To test this hypothesis, HS and LS lines were incubated for 5 h in the presence of APC pulsed with graded concentrations of peptide Ag or titrated amounts of immobilized anti-CD3 Ab. The amount of TCR present on the cell surface was then assessed by staining with a V-specific Ab that recognizes the transgenic TCR. Surprisingly, we found that the concentration of either peptide Ag (Fig. 4A) or anti-CD3 Ab (Fig. 4B) required for TCR internalization correlated with the peptide requirement for CTL activation. Approximately 50% of the TCR was internalized in HS lines following stimulation with APC pulsed with 10^-9 M peptide or 0.16 µg/ml of immobilized anti-CD3 Ab. LS lines require 2 logs more peptide or 3.5-fold more 2C11 to induce equivalent levels of internalization. Thus, CTL with the LS phenotype appear to require more TCR to be engaged than HS CTL to transduce sufficient signaling to achieve activation. These data would support a model in which the differences between the lines in the concentration of peptide required for activation or effector function is the result of differences in the ability of the TCR to transduce signals.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. TCR internalization correlates with the requirement for peptide Ag. A, Dose-dependent TCR down-regulation following Ag exposure. LCMV peptide-pulsed EL4s were cultured with CTL of HS (•) or LS () phenotypes for 5 h. Following Ag exposure, surface expression of TCR was determined by staining CTL for V8 expression and analysis by flow cytometry. B, TCR expression following cross-linking by anti-CD3 Ab. CTL were incubated with plate-bound anti-CD3 Ab for 5 h. Following incubation, TCR surface expression was measured as in A.

HS and LS CTL lines express similar levels of CD8, but differ in their expression of CD8

It has previously been shown that varying the expression of CD8 can modulate the requirement for peptide/MHC (4, 15, 16, 17). To determine whether we had selected for CTL with differences in the level of CD8, we tested the lines for the expression of CD8 and CD8. Flow cytometric analyses were performed on the same day as the data shown in Figs. 2B, 3, 4, and 6. Interestingly, although the expression of CD8 was similar between HS and LS lines (Fig. 5A), the HS line expressed significantly more (1.6-fold) CD8 (Fig. 5B). The increased expression of CD8 in HS lines was remarkably reproducible, with the CD8/CD8 ratios consistently ranging from 1.5 to 1.7 in five lines analyzed. Furthermore, the ratio of CD8/CD8 in the LS lines was always higher when compared with HS lines. In three LS lines generated, the ratio of CD8/CD8 ranged from 2.5 to 3.7, differing significantly from the CD8/CD8 ratios expressed by five HS lines (1.5–1.7). Thus the differential expression of the CD8 chains is highly reproducible. CD8 can exist as a homodimer (CD8) or a heterodimer (CD8), whereas CD8 can only reach the surface as a heterodimer (18). These data demonstrate that CD8 molecules expressed by HS lines are largely heterodimers, whereas LS lines express significantly more homodimers. The possible functional consequences of increased CD8 expression will be discussed below.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. HS and LS CTL lines express similar levels of CD8, but differ in their expression of CD8. Six days post stimulation, HS (dotted lines) and LS (solid lines) CTL were analyzed for surface expression of CD8 and CD8. This analysis was performed on the same day post stimulation as the data in Figs. 2B, 3, 4, and 6. Equivalent expression of CD8 coupled with the disparate expression of CD8 between the lines indicates that HS lines express a higher ratio of CD8/CD8 compared with LS lines.

In addition to measuring the degree of TCR internalization following CD3 engagement, the down-regulation of CD8 was determined by flow cytometric analysis following incubation with titrated concentrations of anti-CD3 Ab. CTL lines of HS and LS phenotypes were treated in the same manner as in Fig. 4B, and CD8 surface expression was measured in combination with TCR expression at each dose of 2C11 using a FITC-conjugated anti-CD8 Ab. Because CD8 is only expressed on the surface in combination with CD8, the CD8 Ab used to measure CD8 down-regulation identifies both CD8 and CD8 isoforms. The data in Fig. 6A are presented as the ratio of CD8 to TCR on the surface of HS and LS CTL lines following CD3 engagement. Thus if TCR and CD8 are internalized at the same rate, the ratio will remain constant. However, if the TCR is selectively internalized while CD8 remains at the cell surface, the ratio will increase. Clearly, as CD3 cross-linking is increased, the internalization of CD8 relative to TCR is diminished in LS cells, while HS cells maintain a nearly constant ratio of CD8/TCR internalization at each concentration of anti-CD3 Ab. Importantly, CD8 internalization occurs in the absence of engagement with peptide MHC complexes, supporting a mechanism in which the localization of CD8 relative to the TCR complex increases the sensitivity to TCR cross-linking. Given the differences in CD8 internalization and the differences in CD8 expression in HS vs LS lines, it remained possible that the CD8 molecules expressing -chains were being selectively internalized following CD3 engagement. To test this hypothesis, expression levels of TCR and CD8 were measured following CD3 engagement. Fig. 6B demonstrates that the ratio of CD8 to TCR at each concentration of anti-CD3 Ab remained nearly constant for both HS and LS CTL lines. These data confirm that CD8 heterodimers are selectively internalized following CD3 engagement, and that the reduced capacity for LS cells to internalize CD8 is a direct result of the decreased expression of CD8 heterodimers. When this study was repeated using another pair of LS and HS lines, similar results were obtained (data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. LS CTL are less efficient at down-regulating CD8 following CD3 engagement due to reduced expression of CD8 heterodimers vs CD8 homodimers. HS (•) or LS () CTL were incubated with titrated concentrations of 2C11 as in Fig. 4. Internalization of the TCR and CD8 or CD8 molecules as a result of CD3 engagement was measured at each concentration of 2C11 by flow cytometric analysis. The expression of these molecules is plotted as the ratio of CD8 (A) or CD8 (B) to TCR detected at each concentration of anti-CD3 Ab.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Although there is a precedent for TCR affinity contributing to the peptide requirements for CTL activation, the data presented in this study have extended our understanding of these requirements to include an additional mechanism. Using a transgenic model system where TCR expression levels and affinity were equivalent and thus could not contribute to any of the observed differences in the sensitivity to peptide Ag, we found that lines with discrete peptide requirements for activation could be generated. In support of these findings, a study by Derby et al. found that in LS and HS lines from nontransgenic mice, TCR affinity as measured by tetramer binding was not necessarily predictive of functional avidity (19). To investigate the mechanism responsible for the differences in the dose-response curves to peptide Ag, CTL were assayed for their functional response in the presence of titrated amounts of anti-CD3 Ab. Importantly, by directly engaging the CD3 complex, any potential contributions from point mutations in the portion of the TCR or functional consequences of CD8 engagement with MHC molecules on APCs were effectively bypassed. Under these activation conditions we found that the CTL lines displayed different requirements for the amount of CD3 engagement needed for activation/effector function.

In nontransgenic CTL lines with HS and LS phenotypes, blocking Abs to CD8 have established that LS cells are much more dependent upon CD8 engagement to achieve activation than HS cells (1, 4). This distinction has been used to define lines functionally as either CD8-dependent (LS) or CD8-independent (HS). In a previous study by Kwan-Lim et al. (20), in which alloreactive T cell hybridomas were analyzed, a correlation between CD8 dependence and sensitivity to CD3 engagement was established. However, in these studies there was an inverse correlation between the expression of CD3 and the responsiveness to anti-CD3 Abs. CD8-independent hybridomas, although more sensitive to CD3 cross-linking, expressed 40–50% less CD3 than CD8-dependent hybridomas. Our results differ from those in the above study in that our CTL lines of HS and LS phenotypes express almost identical levels of CD3 yet still demonstrate differences in the amount of CD3 cross-linking required for activation.

What then is the basis for the differences in the requirement for peptide and anti-CD3 Ab? Although LS and HS lines express equivalent amounts of CD8, LS lines consistently demonstrated a reduced expression of CD8. Thus HS lines have an increased ratio of CD8 heterodimers to CD8 homodimers. This finding suggests that the ratio of CD8 to CD8 is an important determinant in the activation threshold for a CTL. Importantly, the increased sensitivity of HS lines is apparent regardless of whether peptide/MHC or anti-CD3 Ab is used to activate the CTL. Thus the contribution of CD8 to the observed differences in sensitivity of the HS and LS CTL lines is independent of its engagement with MHC.

A study by Anel et al. (21) investigating CD8-independent and -dependent CTL clones found that the CD8-independent CTL were more efficient at recruiting p56^lck following stimulation with anti-CD3 Ab. This finding suggested a possible mechanism to explain the decreased requirement of CTL with high functional avidity for CD8 engagement. The role of CD8 in increasing the efficiency of CD8 coreceptor function is well documented (22, 23, 24). A number of studies have demonstrated the role of the cytoplasmic domain of CD8 in CD8-mediated Lck activity and localization (25, 26, 27). A report by Arcaro et al. (23) demonstrated that heterodimeric CD8 is a more efficient coreceptor than homodimeric CD8. This study found that CD8 is palmitoylated at the cytoplasmic tail of CD8, allowing for the preferential sequestration of CD8 heterodimers into lipid rafts. Furthermore, they showed that CD8-associated p56^lck occurred preferentially in lipid rafts serving to localize the kinase in proximity to raft-associated TCR complexes (23). This results in the efficient phosphorylation of TCR immunoreceptor tyrosine-based activation motifs (ITAMs) found on the cytoplasmic tails of TCR molecules (28, 29).

The linker for activation of T cells (LAT) serves to propagate TCR signaling by binding downstream signaling mediators following phosphorylation by ZAP-70 (30, 31, 32). The amount of CD8-associated LAT was shown to be markedly greater in CD8⁺ T cells than in CD8^- T cells (27), further supporting a role for CD8-mediated sensitivity to CD3 cross-linking. The increased expression of CD8 by our HS CTL lines may confer an increased ability to recruit p56^lck or LAT to the TCR/CD3 complex resulting in increased sensitivity to anti-CD3 Ab.

A requirement for dimerization or trimerization of the TCR for efficient CTL activation to occur has been reported (33). A study by Fahmy et al. (34) has proposed that activation-induced membrane changes in TCR avidity for peptide/MHC complexes between naive and activated T cells could increase the sensitivity of T cells to peptide Ag. The rearrangement of the TCR within the membrane can facilitate multimeric binding of TCR to surface peptide/MHC complexes on APCs. It is possible that our LS lines are less efficient at rearranging the TCR within the membrane, translating functionally into a decreased sensitivity to peptide Ag. Oligomerization of TCR may be controlled by the recruitment of TCR/CD3 complexes into detergent-insoluble membranes (DIMs) or "rafts" following TCR engagement (35). Therefore, it is possible that the increased sensitivity to CD3 cross-linking seen in HS cells is due in part to more efficient localization of TCR molecules into lipid rafts as a result of increased CD8 expression and appropriate p56^lck localization. There is also evidence that TCR-mediated signaling can be enhanced by the extracellular interaction of CD8 with the TCR/CD3 complex (22). Capping studies have shown that anti-CD8 Abs are significantly more efficient at inducing cocapping of the TCR when compared with anti-CD8 Abs (26). Importantly, these studies demonstrate an interaction between CD8 and TCR that occurs independent of their binding to MHC and indicates that this association is stronger for CD8 heterodimers than for CD8 homodimers. Our data support this observation by demonstrating a selective co-internalization of CD8 molecules with the TCR following CD3 engagement.

The data presented in this report significantly change the way one can envision the peptide requirement of an individual CTL. Previously it has been postulated that the control of functional avidity was solely the result of TCR affinity. Thus the requirement for an increased amount of peptide Ag by LS CTL was a reflection of the inefficient interaction of a low affinity TCR with peptide/MHC. An underlying assumption of this model is that all CTL require the same number of TCR engagement events to achieve activation and that depending on the TCR affinity individual clones would need different amounts of peptide to achieve this threshold. Our data would expand this model to include CTL with differences in the threshold level of TCR engagement required to become activated. This difference in threshold may be mediated by disparate expression levels of CD8 heterodimers vs CD8 homodimers in HS and LS CTL. It is thought that the heterodimeric form of CD8 comprises the vast majority of CD8 expressed by CTL. The studies presented herein are the first to show a correlation between the sensitivity of a CTL to peptide Ag and the form of the CD8 molecule expressed by the cell. These data suggest that the amount of peptide required for activation of an individual CTL may be determined by the ratio of CD8 heterodimers vs homodimers expressed. Our findings support a model in which a LS CTL clone could have a TCR that binds peptide/MHC as efficiently as a HS clone, but it would require significantly higher numbers of TCR engagement events to become activated, perhaps as a result of a relatively high CD8 homodimer to CD8 heterodimer ratio. The higher requirement for TCR engagement would result in the necessity for higher levels of peptide/MHC complexes on the APC. Studies are underway to further elucidate the mechanism responsible for the differences observed among the lines in their requirement for TCR engagement, including the role that CD8 serves in the absence of CD8 engagement. Insights gained from these studies further our understanding of the control of CTL activation and may provide new and important information for the design of vaccines and immunomodulatory therapies.

	Acknowledgments

 
We thank Drs. Jay Berzofsky, Michael Derby, Ted Hansen, and Janet Connolly for helpful discussions, and Drs. Steven Mizel and Doug Lyles for careful reading of this manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant AI43591 (to M.A.A.-M.).

² Address correspondence and reprint requests to Dr. Martha A. Alexander-Miller, Department of Microbiology and Immunology, Room 5108, Gray Building, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address: marthaam{at}wfubmc.edu

³ Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; RAG2, recombinase-activating gene 2; HS, highly sensitive; LS, less sensitive; LAT, linker for activation of T cells.

Received for publication May 1, 2001. Accepted for publication July 3, 2001.

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