The CD8 coreceptor on T cells has two functions. Namely, CD8 acts to stabilize the binding of the T-cell receptor (TCR) to the peptide-MHC complex while localizing p56lck (lck) to the TCR/CD3 complex to facilitate early signaling events. Although both functions may be critical for efficient activation of a CTL, little is known about how the structural versus signaling roles of CD8, together with the relative strength of the TCR, influences T-cell function. We have addressed these issues by introducing full-length and truncated versions of the CD8α and CD8β chains into CD8 Jurkat cell clones expressing cloned TCRs with known antigen specificity and relative affinities. Using a combination of antigen recognition and tetramer-binding assays, we find that the intracellular lck-binding domain of CD8 is critical for enhanced T-cell activation regardless of the relative strength of the TCR. In contrast, the extracellular domain of CD8 seems to be critical for TCRs with lower affinity but not those with higher affinity. Based on our results, we conclude that there are different requirements for CD8 to enhance T-cell function depending on the strength of its TCR. (Cancer Res 2006; 66(23): 11455-61)

The T-cell coreceptor CD8 is known to bind class I molecules directly and to be critical for development of CD8+ T cells (1, 2). It is generally accepted that CD8 binding to MHC helps stabilize the T-cell receptor (TCR)/peptide MHC (pMHC) complex and localizes the CD8-associated protein tyrosine kinase lck to the CD3 complex, which aids in initiating the TCR signaling cascade by phosphorylating the ζ chain of the TCR-associated CD3 complex (1, 35). However, the influence of CD8 on the function of T cells expressing high- versus low-affinity TCRs is poorly understood. We and others have speculated that CD8 expression is required only for those TCRs with lower affinity, whereas CD8 expression is not required for T cells expressing higher-affinity TCRs (68). Based on these studies, the contribution of CD8 to TCR/pMHC stability seems to be more important than colocalizing lck to the CD3 complex for T-cell activation.

CD8 is encoded by two distinct genes, CD8α and CD8β, and is expressed on the surface of T cells primarily as a CD8αβ heterodimer (9). The Ig domain of CD8 binds to the conserved α3 domain of MHC class I molecules, whereas the cytoplasmic tail of CD8 binds the Src kinase, lck (10, 11). CD8α recruits signaling components to the cytoplasmic side of the TCR/pMHC interaction (12) and provides the majority of the binding energy in the CD8 heterodimer (13). The tail of the CD8β contains 19 residues and plays a role in positive selection; however, the importance of CD8β for the activation of T cells is not clearly defined. It has been shown that the globular heads of murine CD8αα and CD8αβ bind to MHC with similar affinity, and cell-to-cell adhesion assays have shown that CD8αα and CD8αβ mediate adherence to pMHC equally (14, 15). Despite these data, heterodimeric CD8αβ has consistently been shown to be a better coreceptor than homodimeric CD8αα and correlates with enhanced sensitivity to peptide antigen (16). This may be due to the fact that CD8β is palmitoylated at the cytoplasmic tail and has been shown to induce higher lck activation in lipid rafts (17). Other studies have shown that the glycosylation of CD8β differs between immature and mature thymocytes and that the extent of glycosylation affects its binding to MHC class I (18, 19). Together, these data suggests a distinct role for CD8β, but there is considerable controversy about the critical domains of CD8β and by what mechanism CD8β affects coreceptor function (20).

Studies to determine whether CD8 functions primarily to facilitate TCR binding and stabilization to pMHC, to enhance signal transduction, or a combination both, have yielded conflicting results. TCR affinity has been shown to play a significant role in determining the sensitivity of a TCR to a peptide antigen. It has been shown that some TCRs bind pMHC despite the presence of anti-CD8 antibodies or mutations in the CD8-binding site (21), whereas other TCRs require CD8 stabilization to allow sufficient time for TCR/pMHC interactions (22, 23). Other studies suggest that although CD8 engagement is not necessary for pMHC binding to the TCR, full activation of the T cell does not occur in the absence of CD8 ligation (24). A possible explanation for the discrepancies between various experimental systems is that CD8 coreceptor dependency may vary with the affinity of the TCR for its pMHC ligand (16, 25). Strong agonist-peptide MHC complexes activate T cells without the involvement of CD8-pMHC interaction, whereas for weak ones, this interaction is necessary (26). Analysis using pMHC tetramers have found a correlation between tetramer binding, which is generally accepted as a measure of TCR affinity, and the amount of antigen required to elicit a functional response (2729). However, it has also been shown that some CTL clones generated from tetramer high populations were incapable of recognizing tumor cells efficiently, whereas some tetramer low sorted CTL clones could lyse their tumor targets (27). We recently reported that CD8 can be required for tetramer binding to TCRs that exhibit CD8-independent recognition of tumor cells (29, 30). Thus, factors other than TCR affinity can be important in determining the sensitivity to peptide antigen, and the CD8 coreceptor may play an important role in both functions.

To better define how the cytoplasmic and extracellular portions of CD8 contribute to antigen recognition by T cells, we created full-length and truncated versions of these molecules, which lack the lck-binding domain and introduced them into Jurkat clones expressing high- or low-affinity–cloned TCRs reactive with known melanoma antigens. Here, we show that the stabilization and signaling properties of the CD8 coreceptor are separate entities. Namely, although the intracellular signaling domain significantly enhances sensitivity to peptide antigen as well as tumor cell recognition, it does not contribute to tetramer binding. Furthermore, depending on the relative affinity of the TCR, TCR/pMHC stabilization afforded by the CD8 coreceptor can contribute to T-cell function.

Cells. Cell lines were purchased from American Type Culture Collection (Manassas, VA) unless otherwise noted. Jurkat cells are a CD8-negative human T-cell lymphoma and T2 cells are a Tap-1−/− human HLA-A*0201 B/T lymphoma. Melanoma cell lines, mel 1300 and mel 624, are HLA-A2 positive, whereas mel 888 is HLA-A2 negative (31). All of these cells were maintained in human complete medium, which consisted of RPMI 1640 (Mediatech, Herndon, VA) supplemented with 10% FCS (Gemini Bio-Products, West Sacramento, CA) and 100 units/mL penicillin (Mediatech), 100 μg/mL streptomycin (Mediatech), and 2.92 mg/mL l-glutamine (Mediatech). 293GP retroviral producer cells were maintained in DMEM (Mediatech) supplemented as above. The original TIL 5 and TIL 1383I lines were established from surgical specimens obtained from melanoma patients undergoing immunotherapy in the Surgical Branch, National Cancer Institute. T-cell clones were grown in X-Vivo 15 (Cambrex, Walkersville, MD) medium supplemented with 10% heat-inactivated human AB serum (Valley Biomedical, Winchester, VA), 100 units/mL penicillin (Invitrogen, Carlsbad, CA), 100 μg/mL streptomycin (Invitrogen), 2.92 mg/mL l-glutamine (Invitrogen), and 6,000 IU/mL recombinant human interleukin 2 (IL-2; Chiron, Emeryville, CA). Because the parental TIL 5 clone is no longer available for analysis, another MART-127-35–reactive T-cell clone was used as a positive control for HLA-A*0201 MART-127-35 tetramer staining.

Reagents and antibodies. The HLA-A*0201–binding peptides MART-127-35 (AAGIGILTV) and tyrosinase368-376 (YMDGTMSQV) were synthesized at Macromolecular Resources (Fort Collins, CO) and purified by reversed-phase high-performance liquid chromatography (HPLC). Purity was assessed by analytic HPLC and was determined to be >99%. Anti-human CD4-FITC and anti-human CD8α-FITC were purchased from BD Biosciences (San Diego, CA). Anti-human CD8 β-phycoerythrin was purchased from Immunotech (Marseilles, France). Anti-human Vβ12-FITC was purchased from Pierce Biotechnology (Rockford, IL). HLA-A*0201 tetramers labeled with phycoerythrin and containing the MART-127-35 and tyrosinase368-376 peptide were obtained from Beckman Coulter (Fullerton, CA).

Gene identification, cloning, and analysis. The identification and cloning of TCR α and β chains from the TIL 5 and TIL 1383I have been described elsewhere (6, 8). The cloning of the CD8α and CD8β chains was done by reverse transcription-PCR using CD8α- and CD8β-specific primers for the full-length and truncated versions of each chain. Total RNA was isolated from 5 × 106 T cells using RNAeasy kits (Qiagen, Valencia, CA) according to the instructions of the manufacturer. First-strand cDNA was prepared from 1 μg total RNA using Superscript II reverse transciptase (Invitrogen) and oligo(dT)12-18 (Invitrogen). Ten nanograms of cDNA were PCR amplified in a 50 μL reaction consisting of 1× PCR buffer (Invitrogen), 1.5 mmol/L MgCl2 (Invitrogen), 200 μmol/L deoxynucleotide triphosphate (Invitrogen), 400 nmol/L CD8α- or CD8β-specific forward primer, 400 nmol/L CD8α or CD8β chain intracellular region specific or CD8α or CD8β transmembrane reverse primers, and 2.5 units Taq DNA polymerase (Continental Lab Products, San Diego, CA). PCR amplification was done using a MJ Research (Watertown, MA) thermocycler under the following conditions: 5 minutes at 92°C (one cycle) followed by 30 seconds at 92°C, 30 seconds at 58°C, and 1 minute at 72°C (35 cycles), followed by 5 minutes at 72°C (one cycle). This allowed for the amplification of either full-length CD8α and CDβ or truncated CD8α and CDβ (α′ or β′). The resulting PCR products were separated on 1% agarose gels containing ethidium bromide (Continental Lab Products) and were visualized under UV light. The products of each PCR reaction were ligated into the pCR 2.1 TA cloning vector (Invitrogen) and transformed into Escherichia coli DH5α-competent cells (Invitrogen). Bacterial clones were screened by PCR using the specific CD8α and CD8β primers to identify clones containing inserts of the predicted size. DNA was isolated from those clones and their inserts were sequenced by cycle sequencing using BigDye Terminator Cycle Sequencing kits (Applied Biosystems, Foster City, CA) and analyzed on an ABI Prism 310 Genetic Analyzer (Applied Biosystems) to ensure there were no PCR errors in the sequence.

Retroviral vector construction. The SAMEN cytomegalovirus (CMV)/SRα retroviral vector was designed specifically for introducing TCR and accessory molecules into alternate T cells. Construction of TIL 1383I and TIL 5 TCR retroviral constructs has been described previously (7, 8). Briefly, the 5′ long terminal repeat (LTR) in the SAMEN SRα backbone was replaced with a hybrid LTR consisting of the human CMV enhancer and promoter fused to the Moloney murine leukemia virus 5′ LTR. This modification permits production of retroviral supernatants by transiently transfecting 293 GP cells (8). Other key elements of the SAMEN CMV/SRα vector include an internal SRα promoter to permit the expression of multiple genes, unique SalI and XhoI restriction sites for ease of inserting TCR chains, and an internal ribosome entry site/neor cassette for G418 selection. A rapid ligation strategy was used to subclone the CD8α and CD8β chain genes into SAMEN CMV SRα. CD8α and CD8α′ genes were excised from pCR2.1 with XhoI and ligated into the SalI restriction site in SAMEN CMV/SRα using a mixture of T4 DNA ligase and SalI restriction endonuclease. The resulting SalI/XhoI hybrid sites are resistant to digestion by SalI and XhoI. Ligation reactions were redigested with SalI, resulting in linearization of plasmids not containing the CD8α chain inserts, allowing for enrichment of recombinant clones. The ligation reactions were cloned into E. coli DH5α-competent cells (Invitrogen), and bacterial clones were screened by PCR using primers that flanked the cloning sites in SAMEN CMV/SRα. The DNA sequence of clones containing inserts was determined using BigDye Terminator Cycle Sequencing kits and analyzed using an ABI Prism 310 Genetic Analyzer (Perkin-Elmer/ABI, Foster City, CA) to ensure that CD8 gene inserts were in the proper orientation. This method was repeated to insert the CD8β chains into the retroviral vector by ligating a SalI fragment containing the β-chain into the XhoI site of SAMEN CMV/SRα to create two CD8 retroviruses, CD8αβ and CD8α′β′ (Fig. 1).

Figure 1.

Structure of the wild-type and truncated CD8. Retroviral vectors encoding CD8 were used to transduce Jurkat cells to express the wild-type or truncated CD8αβ heterodimers. A, the CD8 retroviruses were created by inserting the CD8α and CD8β genes into the SAMEN CMV/SRα retroviral vector. The vector is composed of a Moloney murine leukemia virus backbone. The 5′ LTR has been modified by replacing the R and U5 segments with a CMV IE promoter enhancer to promote high-level transient expression in 293GP producer cells. Other key elements include the ψ+ packaging signal, splice donor and splice acceptor, an internal SRα promoter to allow expression of a second gene, CD8β and CD8α chains inserted into unique SalI and XhoI restriction sites, respectively, and an internal ribosomal entry site (IRES)/neor cassette for expression of a geneticin resistance gene. B, a schematic illustration of the CD8 molecules used in these studies. CD8αβ depicts the wild-type genes of the heterodimer, including the lck-binding domain on the intracellular α chain. CD8α′β′ depicts the truncated copies of both the α and β heterodimer.

Figure 1.

Structure of the wild-type and truncated CD8. Retroviral vectors encoding CD8 were used to transduce Jurkat cells to express the wild-type or truncated CD8αβ heterodimers. A, the CD8 retroviruses were created by inserting the CD8α and CD8β genes into the SAMEN CMV/SRα retroviral vector. The vector is composed of a Moloney murine leukemia virus backbone. The 5′ LTR has been modified by replacing the R and U5 segments with a CMV IE promoter enhancer to promote high-level transient expression in 293GP producer cells. Other key elements include the ψ+ packaging signal, splice donor and splice acceptor, an internal SRα promoter to allow expression of a second gene, CD8β and CD8α chains inserted into unique SalI and XhoI restriction sites, respectively, and an internal ribosomal entry site (IRES)/neor cassette for expression of a geneticin resistance gene. B, a schematic illustration of the CD8 molecules used in these studies. CD8αβ depicts the wild-type genes of the heterodimer, including the lck-binding domain on the intracellular α chain. CD8α′β′ depicts the truncated copies of both the α and β heterodimer.

Close modal

Generation of retroviral supernatants. One hundred millimeter tissue culture plates were coated with 0.2% type B bovine skin gelatin (Sigma) in HBSS for 15 minutes at room temperature. 293 GP cells were seeded onto coated plates at sufficient density to provide 70% confluency after 24 hours (∼3 × 106). Monolayers were rinsed thrice with PBS and transiently cotransfected using LipofectAMINE and PLUS reagents (Life Technologies), 3 μg of the retroviral plasmid containing the CD8 genes, and 3 μg vesicular stomatitis virus envelope gene in 6.0 mL serum-free DMEM. Following a 3-hour incubation, 10 mL DMEM with 20% FCS was added to the flasks, and cells were incubated at 37°C. Medium was discarded after 24 hours and replaced with 10 mL human complete medium. Retroviral supernatants were collected after 24 hours, replaced with medium, and collected again after an additional 24 hours. Retroviral supernatants were either used immediately or frozen at −70°C for later use.

Retroviral transduction. Fresh retroviral supernatants were supplemented with 8 μg/mL polybrene (Sigma) and filter sterilized. Jurkat cells were suspended at 1 × 106/mL in retroviral supernatant. One milliliter of cells per supernatant was added to each well of 24-well tissue culture plate, and the plates were centrifuged at 1,000 × g for 90 minutes at 32°C. Plates were returned to the incubator and, after 4 hours, 1 mL fresh RPMI was added to each well. Transduced cells were incubated overnight, and this procedure was repeated the next day with fresh supernatant as described above.

Antigen recognition assay. Retrovirally transduced Jurkat cells were tested for reactivity to tumor antigens in cytokine release assays. Tumor cells or T2 cells preincubated for 2 hours with 10 μg/mL peptides were washed twice with PBS and then added to effector cells at a 1:1 ratio in a total volume of 200 μL human complete medium per well of a 96-well, U-bottomed tissue culture plate. Actual numbers were 2 × 105 cells per well. Cocultures were incubated at 37°C in a humidified CO2 incubator for 24 hours. Supernatants were harvested, and the amount of human IL-2 (R&D Systems, Minneapolis, MN) released by Jurkat cells was measured by ELISA (R&D Systems).

Influence of CD8 on tetramer binding by TCR-transduced Jurkat clones. We have previously shown that the TIL 5 and TIL 1383I TCRs can transfer HLA-A2–restricted MART-127-35 and tyrosinase368-376 reactivity, respectively, to Jurkat cells, peripheral blood lymphocyte–derived T cells, and established T-cell lines and clones (68, 32). Despite their strong reactivity toward the peptide antigen, the TIL 5 TCR–transduced cells were unable to recognize melanoma tumor cells without CD8 expression, whereas TIL 1383I TCR–transduced cells could (7, 8). Based on the dependency of CD8 for tumor recognition, we consider the TIL 1383I TCR to have higher relative affinity for tumor antigen than the TIL 5 TCR. We have also found that regardless of their ability to recognize tumor cells, neither the TIL 5 nor TIL 1383I TCR–transduced Jurkat cells were capable of binding MART-127-35– or tyrosinase368-376 peptide–loaded HLA-A2 tetramers (29). Therefore, the ability of a TCR to recognize tumor antigen did not always correlate with its affinity for the tetramer. This underscores the importance of defining the contributions of CD8 on the TCR affinity for tetramer and avidity for antigen.

To better define the role of CD8 in TCR/pMHC interactions and tumor cell recognition, we transduced Jurkat clones expressing the TIL 5 (clone 22) or TIL 1383I (clone C4) TCRs with retroviral vectors encoding wild-type CD8αβ or CD8α′β′ (Fig. 1). Jurkat cells were used as a readout for T-cell activation and function in these studies because they are easily gene modified (6, 8, 30) and their signaling properties have been well characterized (33). As shown in Fig. 2, the resulting Jurkat cells expressed high levels of CD8αβ or CD8α′β′ heterodimers. CD8α′β′ lack cytoplasmic tails and therefore cannot colocalize lck to the CD3 complex; however, its intact extracellular α domain is still capable of binding to MHC I. The expression of CD8αβ or CD8α′β′ on TIL 1383I TCR Jurkat cells had little effect on TCR expression because Vβ12 levels were similar in the untransduced, CD8αβ-transduced, or CD8α′β′-transduced Jurkat clone C4 cells (Fig. 3). Similar analysis could not be done on TIL 5 TCR Jurkat cells because there are no antibodies available that are capable of staining the TIL 5 TCR (6).

Figure 2.

Expression of CD8αβ and CD8α′β′ on TCR-transduced Jurkat clones. TIL 1383I TCR Jurkat clone C4 cells and TIL 5 TCR Jurkat clone 22 cells were transduced with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector. Stable transductants were stained for expression of either CD8α-FITC or CD8 β-phycoerythrin and fluorescence was quantified by flow cytometry. Histograms, relative log fluorescence of 104 viable cells.

Figure 2.

Expression of CD8αβ and CD8α′β′ on TCR-transduced Jurkat clones. TIL 1383I TCR Jurkat clone C4 cells and TIL 5 TCR Jurkat clone 22 cells were transduced with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector. Stable transductants were stained for expression of either CD8α-FITC or CD8 β-phycoerythrin and fluorescence was quantified by flow cytometry. Histograms, relative log fluorescence of 104 viable cells.

Close modal
Figure 3.

Relationship between CD8 and TCR expression on TIL 1383I TCR Jurkat cells. The levels of CD8 and Vβ12 expression were measured on TIL 1383I TCR Jurkat cells to ensure that the retroviral transduction did not significantly alter the expression of the TIL 1383I TCR. Untransduced clone C4 cells and clone C4 cells transduced with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector were stained for expression of CD8 (anti-CD8α-phycoerythrin) or the TIL 1383I TCR (anti-Vβ12-FITC). Fluorescence staining was quantified by flow cytometry. Histograms, relative log fluorescence of 104 viable cells.

Figure 3.

Relationship between CD8 and TCR expression on TIL 1383I TCR Jurkat cells. The levels of CD8 and Vβ12 expression were measured on TIL 1383I TCR Jurkat cells to ensure that the retroviral transduction did not significantly alter the expression of the TIL 1383I TCR. Untransduced clone C4 cells and clone C4 cells transduced with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector were stained for expression of CD8 (anti-CD8α-phycoerythrin) or the TIL 1383I TCR (anti-Vβ12-FITC). Fluorescence staining was quantified by flow cytometry. Histograms, relative log fluorescence of 104 viable cells.

Close modal

We have found that the introduction of the CD8αβ molecule into TIL 1383I TCR Jurkat cells resulted in tetramer binding, whereas cells transduced with the empty vector failed to bind tetramers (Fig. 4A). CD8α′β′ expressing TIL 1383I TCR Jurkat cells could also bind tetramers, indicating that the cytoplasmic tails had no effect on the ability of CD8 to stabilize the TCR/pMHC complex (Fig. 4A). Although the expression of CD8 facilitated tetramer binding to TIL 1383I TCR-expressing cells, TIL 5 TCR Jurkat cells with or without CD8 failed to bind tetramers (data not shown). These results indicate that CD8 can facilitate tetramer binding to some, but not all, TCRs.

Figure 4.

Influence of CD8 expression on tetramer staining and antigen recognition by TIL 1383I TCR Jurkat cells. The influence of CD8 expression on Jurkat cells expressing the TIL 1383I TCR was determined. A, the influence of CD8 expression on the stability of TCR/pMHC complexes was determined. Untransduced clone C4 cells (shaded curve) and clone C4 cells transduced (open curves) with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector were stained HLA-A2/tyrosinase368-376 phycoerythrin tetramers. Fluorescence staining was quantified by flow cytometry. Histograms, relative log fluorescence of 104 viable cells. B, the influence of CD8 expression on antigen recognition was determined. Clone C4 cells transduced with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector were cocultured overnight with peptide-loaded T2 cells (striped columns) or melanoma cell lines (filled columns). Peptides used were MART-127-35 and tyrosinase368-376. Melanoma cells used were mel 1300 (HLA-A2+), mel 624 (HLA-A2+), and mel 888 (HLA-A2). The antigen recognition was assessed by the release of IL-2. The amount of cytokine released was measured by ELISA. Columns, average of triplicate wells. Representative of three replicate experiments. C, the influence of CD8 expression on the functional avidity or sensitivity of TIL 1383I TCR Jurkat cells to antigen stimulation was determined. Clone C4 cells transduced with the empty (•), CD8αβ-containing (▴), or CD8α′β′-containing (▾) SAMEN CMV/SRα retroviral vector were cocultured overnight with T2 cells loaded with six different concentrations of tyrosinase368-376 peptide. The antigen recognition was assessed by the release of IL-2. The amount of cytokine released was measured by ELISA. There was no IL-2 release (<32 pg/mL, which was below the lowest value on the standard curve) against an irrelevant MART-127-35 control peptide. Points, average of triplicate wells. Representative of three independent experiments.

Figure 4.

Influence of CD8 expression on tetramer staining and antigen recognition by TIL 1383I TCR Jurkat cells. The influence of CD8 expression on Jurkat cells expressing the TIL 1383I TCR was determined. A, the influence of CD8 expression on the stability of TCR/pMHC complexes was determined. Untransduced clone C4 cells (shaded curve) and clone C4 cells transduced (open curves) with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector were stained HLA-A2/tyrosinase368-376 phycoerythrin tetramers. Fluorescence staining was quantified by flow cytometry. Histograms, relative log fluorescence of 104 viable cells. B, the influence of CD8 expression on antigen recognition was determined. Clone C4 cells transduced with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector were cocultured overnight with peptide-loaded T2 cells (striped columns) or melanoma cell lines (filled columns). Peptides used were MART-127-35 and tyrosinase368-376. Melanoma cells used were mel 1300 (HLA-A2+), mel 624 (HLA-A2+), and mel 888 (HLA-A2). The antigen recognition was assessed by the release of IL-2. The amount of cytokine released was measured by ELISA. Columns, average of triplicate wells. Representative of three replicate experiments. C, the influence of CD8 expression on the functional avidity or sensitivity of TIL 1383I TCR Jurkat cells to antigen stimulation was determined. Clone C4 cells transduced with the empty (•), CD8αβ-containing (▴), or CD8α′β′-containing (▾) SAMEN CMV/SRα retroviral vector were cocultured overnight with T2 cells loaded with six different concentrations of tyrosinase368-376 peptide. The antigen recognition was assessed by the release of IL-2. The amount of cytokine released was measured by ELISA. There was no IL-2 release (<32 pg/mL, which was below the lowest value on the standard curve) against an irrelevant MART-127-35 control peptide. Points, average of triplicate wells. Representative of three independent experiments.

Close modal

Influence of CD8 on antigen recognition by TCR-transduced Jurkat clones. In addition to TCR/pMHC stabilization, we analyzed the effect of the structural domains of CD8 on antigen recognition by TCR-transduced Jurkat cells. To assess the capacity of the different forms of CD8 to affect antigen recognition, we did cytokine release assays using our panel of TCR-transduced Jurkat cells. Each Jurkat clone transduced with the empty vector, CD8αβ, or CD8α′β′ was cocultured with T2 cells loaded with the MART-127-35 or tyrosinase368-376 peptide, HLA-A2–positive tumor cell lines (mel 1300 and mel 624), or an HLA-A2–negative tumor cell line (mel 888). Responder and stimulator cells were cocultured overnight. The next day, supernatants were removed and the amount of IL-2 was measured by ELISA.

The addition of CD8αβ consistently showed the greatest increase in IL-2 production when compared with empty vector controls for both TIL 5 (Fig. 5A) and TIL 1383I (Fig. 4B) TCR Jurkat cells. However, the TIL 5 TCR Jurkat cells, which previously only recognized peptide-loaded targets (6), were now able to recognize mel 1300 cells but not mel 624 when expressing CD8αβ heterodimers. The most likely explanation for the differential recognition of the melanomas by CD8αβ heterodimers in TIL 5 TCR Jurkat cells is that mel 1300 has higher levels of HLA-A2, MART-1, and tyrosinase expression than mel 624 (31). These results indicate that CD8αβ heterodimers can enhance but are not required for recognition of peptide-loaded targets and tumor cells by Jurkat cells expressing a TCR with high relative affinity (TIL 1383I). In contrast, CD8 expression is essential for tumor cell recognition by Jurkat cells expressing a TCR with low relative affinity (TIL 5).

Figure 5.

Influence of CD8 expression on antigen recognition by TIL 5 TCR Jurkat cells. The influence of CD8 expression on Jurkat cells expressing the TIL 5TCR was determined. A, the influence of CD8 expression on antigen recognition was determined. Clone 22 cells transduced with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector were cocultured overnight with peptide-loaded T2 cells (striped columns) or melanoma cell lines (filled columns). Peptides used were MART-127-35 and tyrosinase368-376. Melanoma cells used were mel 1300 (HLA-A2+), mel 624 (HLA-A2+), and mel 888 (HLA-A2). The antigen recognition was assessed by the release of IL-2. The amount of cytokine released was measured by ELISA. Columns, average of triplicate wells. Representative of three replicate experiments. B, the influence of CD8 expression on the functional avidity or sensitivity of TIL 5 TCR Jurkat cells to antigen stimulation was determined. Clone 22 cells transduced with the empty (•), CD8αβ-containing (▴), or CD8α′β′-containing (▾) SAMEN CMV/SRα retroviral vector were cocultured overnight with T2 cells loaded with five different concentrations of MART-127-35 peptide. The antigen recognition was assessed by the release of IL-2. The amount of cytokine released was measured by ELISA. There was no IL-2 release (<32 pg/mL, which was below the lowest value on the standard curve) against an irrelevant tyrosinase368-376 control peptide. Points, average of triplicate wells. Representative of three independent experiments.

Figure 5.

Influence of CD8 expression on antigen recognition by TIL 5 TCR Jurkat cells. The influence of CD8 expression on Jurkat cells expressing the TIL 5TCR was determined. A, the influence of CD8 expression on antigen recognition was determined. Clone 22 cells transduced with the empty, CD8αβ-containing, or CD8α′β′-containing SAMEN CMV/SRα retroviral vector were cocultured overnight with peptide-loaded T2 cells (striped columns) or melanoma cell lines (filled columns). Peptides used were MART-127-35 and tyrosinase368-376. Melanoma cells used were mel 1300 (HLA-A2+), mel 624 (HLA-A2+), and mel 888 (HLA-A2). The antigen recognition was assessed by the release of IL-2. The amount of cytokine released was measured by ELISA. Columns, average of triplicate wells. Representative of three replicate experiments. B, the influence of CD8 expression on the functional avidity or sensitivity of TIL 5 TCR Jurkat cells to antigen stimulation was determined. Clone 22 cells transduced with the empty (•), CD8αβ-containing (▴), or CD8α′β′-containing (▾) SAMEN CMV/SRα retroviral vector were cocultured overnight with T2 cells loaded with five different concentrations of MART-127-35 peptide. The antigen recognition was assessed by the release of IL-2. The amount of cytokine released was measured by ELISA. There was no IL-2 release (<32 pg/mL, which was below the lowest value on the standard curve) against an irrelevant tyrosinase368-376 control peptide. Points, average of triplicate wells. Representative of three independent experiments.

Close modal

The fact that CD8 enhances antigen recognition is consistent with our previous studies of the TIL 5 TCR (6, 7). However, we have speculated that the observed tumor cell recognition by TIL 5 TCR–transduced CD8+ T cells was due to the structural properties of CD8 (6, 7). Based on these results, we considered enhanced TCR affinity or stability of the TCR/pMHC complex to be the key to efficient activation of TCR gene-modified cells (7, 8). Although we have no direct evidence for the TIL 5 TCR, the correlation between tetramer binding to TIL 1383I TCR Jurkat-expressing cells CD8αβ (Fig. 4A) and enhanced recognition of peptide-loaded T2 cells and tumor cells (Fig. 4B) supports this hypothesis.

To directly assess the structural versus the signaling functions of CD8 in antigen recognition by TIL 5 and TIL 1383I TCR Jurkat cells, cells transduced to express the α′β′ form of CD8, which lacks the lck-binding domain, were assayed for recognition of peptide-loaded T2 cells and melanoma cells. As shown in Figs. 4B and 5A, the expression of CD8α′β′ had different effects on IL-2 secretion depending on the TCR expressed. When CD8α′β′ is expressed by TIL 1383I TCR Jurkat cells, the amount of IL-2 released was comparable with that of cells transduced with the empty vector (Fig. 4B). It should be noted that the expression of CD8α′β′ stabilized the TCR/pMHC complex as illustrated by the ability of these cells to bind tetramers (Fig. 4A). Therefore, it is the signaling function, and not the MHC-binding function of CD8, that influences antigen recognition by TIL 1383I TCR–transduced Jurkat cells. In contrast, expression of CD8α′β′ in TIL 5 TCR Jurkat cells led to enhanced IL-2 release relative to cells transduced with the empty vector when stimulated with peptide-loaded T2 cells (Fig. 5A). Like cells expressing the wild-type CD8 molecule, cells expressing CD8α′β′ could recognize mel 1300, whereas cells transduced with the empty vector could not. The amount of IL-2 released by CD8α′β′ expressing TIL 5 TCR Jurkat cells was less than CD8αβ expressing TIL 5 TCR Jurkat cells, indicating that colocalization of lck to the CD3 complex by CD8 was also important for tumor cell recognition by TIL 5 TCR-expressing cells. Based on these results, we conclude that stabilizing the TCR/pMHC complex by CD8αβ heterodimers can increase the efficiency of antigen recognition by TCR-transduced cells, and, depending on the strength of the TCR, CD8 expression is essential for allowing tetramer binding and for tumor cell recognition to occur.

Influence of CD8 on the functional avidity of Jurkat clones. It has been argued that tumor cell recognition is related to the functional avidity or sensitivity of a T-cell to antigen stimulation (34, 35). To determine how the structural versus signaling features of CD8 influence functional avidity, we cocultured our Jurkat clones transduced with the empty vector, CD8αβ, or CD8α′β′ with T2 cells loaded with differing concentration of either the MART-127-35 or the tyrosinase368-376 peptides. Responder and stimulator cells were cocultured overnight, supernatants were removed the next day, and the amount of IL-2 was measured by ELISA. The expression of CD8αβ increased the sensitivity of both TIL 5 (Fig. 5B) and TIL 1383I (Fig. 4C) TCR Jurkat cells to antigen stimulation compared with the empty vector controls (Figs. 4C and 5B). As we have observed with tumor cell recognition, the influence of CD8α′β′ on functional avidity depended on which TCR was expressed. Expression of CD8α′β′ on TIL 1383I TCR Jurkat cells had little effect on their sensitivity to antigen stimulation (Fig. 4C). These cells were only slightly more sensitive to antigen than cells transduced with the empty vector, which required ∼10-fold more peptide to activate the cells than CD8αβ TIL 1383I TCR Jurkat cells. In contrast, expression of CD8α′β′ on TIL 5 TCR Jurkat cells significantly increased their sensitivity to antigen stimulation relative to cells transduced with the empty vector (Fig. 5B). However, the sensitivity of CD8α′β′ TIL 5 TCR Jurkat cells to antigen stimulation was less than CD8αβ TIL 5 TCR Jurkat cells, indicating that the signaling function as well as the MHC-binding capability of CD8 are required for optimum activation of TIL 5 TCR–transduced cells.

Many studies have been aimed at determining the relationships between T-cell function, TCR affinity, and CD8 dependence (3638). Despite these efforts, questions about the relative effect of CD8 remain. For example, we and others have argued that CD8-independent tumor cell recognition is a property of a high-affinity TCR (8, 30, 39, 40). Others argue that T cells that stain with tetramers engineered such that they will not bind CD8 express high-affinity TCRs (4143). In our hands, the two TCRs we have identified that exhibit CD8-independent tumor cell recognition fail to bind tetramers in the absence of CD8 (29, 30). This discrepancy between the formation and stability of TCR/pMHC complexes and T-cell function led to the current study in which we introduced wild-type or truncated CD8 molecules into Jurkat cells expressing well-characterized TCRs. This approach allowed us to separate the structural and signaling features of CD8 to determine which is more important for T-cell function.

Results obtained in this study challenge the hypothesis that increased TCR affinity through the addition of CD8 leads to enhanced T-cell function. Based on the analysis of two different TCRs, we find that it is the affinity of the TCR for their pMHC ligand that dictates its need for CD8. Furthermore, the differences in the CD8α/CD8β ratios between TIL 5 and TIL 1383I TCR–transduced Jurkat cells or the requirement of CD8β do not effect on our results. TIL 5 and TIL 1383I Jurkat cells expressing CD8 are similar to TCR-transduced T-cell clones in their ability to bind tetramers and recognize antigen. The TIL 5 TCR, which has lower affinity, supports the aforementioned hypothesis because TIL 5 TCR Jurkat cells expressing CD8α′β′ have higher functional avidity than cells transduced with the empty vector. This enhancement of functional reactivity translated into the ability of TIL 5 TCR Jurkat cells expressing CD8α′β′ to recognize mel 1300 cells, whereas cells transduced with the empty vector cannot. In contrast, the higher-affinity TIL 1383I TCR does not support this hypothesis. Despite the fact that CD8α′β′ expression allows tetramers to bind to TIL 1383I TCR Jurkat cells, the functional avidity and the amount of IL-2 secreted by these cells when stimulated with tumor cells is comparable regardless of whether or not they express CD8α′β′. These results suggest that enhancing the stability of the TCR/pMHC complex is important for low-affinity TCRs but not for high-affinity TCRs.

By comparing cells expressing CD8αβ versus CD8α′β′ coreceptors, we were able to determine to what extent colocalizing lck to the CD3 complex influenced T-cell activation. Here again, the results obtained were dependent on the TCR. In all cases, the ability of CD8αβ to colocalize lck to the CD3 complex led to enhanced IL-2 secretion. For Jurkat cells expressing the TIL 5 TCR, the signaling function augmented the structural role of CD8 leading to the highest functional avidity and greatest amount of IL-2 secreted when stimulated with peptide-loaded T2 cells or mel 1300 cells. These results indicate that for a lower-affinity TCR, enhancing the signaling potential of the T cells synergizes with stabilizing the TCR/pMHC complex for optimum T-cell activation. For Jurkat cells expressing the TIL 1383I TCR, expression of CD8αβ, but not CD8α′β′, led to increased functional avidity and enhanced IL-2 production when stimulated with peptide-loaded T2 cells or melanoma cells. Therefore, for high-affinity TCRs, enhancing the signaling potential of the T cells outweighs the benefits of further stabilizing the TCR/pMHC complex and leads to better T-cell function.

The emerging picture suggests that coreceptor function in T-cell activation is related to the strength of the TCR/pMHC complex. The data presented here agree with this model but also determines which domains of the coreceptor are most critical for T-cell function. Here, we conclude that the sensitivity and function of T cells to antigen stimulation is dependent on the ability of the intracellular domain of CD8 to localize lck to the CD3 complex and, thus, it is signaling rather than the stabilization of the TCR/p/MHC complex that is responsible for the increase in T-cell activity afforded by CD8. This conclusion is supported by structural studies that indicate that the binding of one molecule is unlikely to alter the affinity for the other (44). We should make it clear that we do not entirely discount the effect of the contribution of the extracellular domain in enhancing the TCR/pMHC interaction. Clearly, the simultaneous binding of the TCR and CD8 by pMHC allows potential for CD8-induced stabilization of the TCR/pMHC interaction. Despite the low affinity of the pMHC/CD8 interaction, any such stabilization could be of extreme biological importance. This is clearly shown by the need for CD8 in normal T cells expressing the TIL 5 and other lower-affinity TCRs (8, 30, 39, 40). When considering strategies to enhance the function of normal CD8+ T cells or TCR-transduced CD8+ T cells for increased therapeutic efficacy, altering the stability of the TCR/pMHC complex would be difficult at best. The implications from our current study is that any means by which the signaling function of a T cell can be enhanced would lead to better T cells regardless of the affinity of the TCR for its pMHC ligand. Strategies to enhance T-cell function by altering their signaling pathways using cytokines and reversing inhibitory signals are currently being explored.

Grant support: NIH grants CA90873, CA111040, and CA102280.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Barber EK, Dasgupta JD, Schlossman SF, Trevillyan JM, Rudd CE. The CD4 and CD8 antigens are coupled to a protein-tyrosine kinase (p56lck) that phosphorylates the CD3 complex.
Proc Natl Acad Sci U S A
1989
;
86
:
3277
–81.
2
Fung-Leung WP, Schilham MW, Rahemtulla A, et al. CD8 is needed for development of cytotoxic T cells but not helper T cells.
Cell
1991
;
65
:
443
–9.
3
Emmrich F, Strittmatter U, Eichmann K. Synergism in the activation of human CD8 T cells by cross-linking the T-cell receptor complex with the CD8 differentiation antigen.
Proc Natl Acad Sci U S A
1986
;
83
:
8298
–302.
4
Veillette A, Bookman MA, Horak EM, Bolen JB. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck.
Cell
1988
;
55
:
301
–8.
5
Salter RD, Benjamin RJ, Wesley PK, et al. A binding site for the T-cell co-receptor CD8 on the α3 domain of HLA-A2.
Nature
1990
;
345
:
41
–6.
6
Cole DJ, Weil DP, Shilyansky J, et al. Characterization of the functional specificity of a cloned T-cell receptor heterodimer recognizing the MART-1 melanoma antigen.
Cancer Res
1995
;
55
:
748
–52.
7
Clay TM, Custer MC, Sachs J, Hwu P, Rosenberg SA, Nishimura MI. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity.
J Immunol
1999
;
163
:
507
–13.
8
Roszkowski JJ, Lyons GE, Kast WM, Yee C, Van Besien K, Nishimura MI. Simultaneous generation of CD8+ and CD4+ melanoma reactive T cells by retroviral mediated TCR gene transfer.
Cancer Res
2005
;
65
:
1570
–6.
9
Ledbetter JA, Seamon WE, Hsu TT, Herzenberg LA. Lyt-2 and Lyt-3 antigens are on two different polypeptide subunits linked by disulfide bonds.
J Exp Med
1981
;
153
:
1503
–16.
10
Salter RD, Norment AM, Chen BP, et al. Polymorphism in the α3 domain of HLA-A molecules affects binding to CD8.
Nature
1989
;
338
:
345
–7.
11
Chalupny NJ, Ledbetter JA, Kavathas P. Association of CD8 with p56lck is required for early T cell signalling events.
EMBO J
1991
;
10
:
1201
–7.
12
Devine L, Sun J, Barr MR, Kavathas PB. Orientation of the Ig domains of CD8αβ relative to MHC class I.
J Immunol
1999
;
162
:
846
–51.
13
Janeway CA, Jr. The T cell receptor as a multicomponent signalling machine: CD4/CD8 coreceptors and CD45 in T cell activation.
Annu Rev Immunol
1992
;
10
:
645
–74.
14
Kern P, Hussey RE, Spoerl R, Reinherz EL, Chang HC. Expression, purification, and functional analysis of murine ectodomain fragments of CD8αα and CD8αβ dimers.
J Biol Chem
1999
;
274
:
27237
–43.
15
Sun J, Kavathas PB. Comparison of the roles of CD8αα and CD8αβ in interaction with MHC class I.
J Immunol
1997
;
159
:
6077
–82.
16
Holler PD, Kranz DM. Quantitative analysis of the contribution of TCR/pepMHC affinity and CD8 to T cell activation.
Immunity
2003
;
18
:
255
–64.
17
Arcaro A, Gregoire C, Boucheron N, et al. Essential role of CD8 palmitoylation in CD8 coreceptor function.
J Immunol
2000
;
165
:
2068
–76.
18
Daniels MA, Devine L, Miller JD, et al. CD8 binding to MHC class I molecules is influenced by T cell maturation and glycosylation.
Immunity
2001
;
15
:
1051
–61.
19
Moody AM, Chui D, Reche PA, Priatel JJ, Marth JD, Reinherz EL. Developmentally regulated glycosylation of the CD8αβ coreceptor stalk modulates ligand binding.
Cell
2001
;
107
:
501
–12.
20
Arcaro A, Gregoire C, Bakker TR, et al. CD8β endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.
J Exp Med
2001
;
194
:
1485
–95.
21
Muller D, Pederson K, Murray R, Frelinger JA. A single amino acid substitution in an MHC class I molecule allows heteroclitic recognition by lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes.
J Immunol
1991
;
147
:
1392
–7.
22
Garcia KC, Scott CA, Brunmark A, et al. CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes.
Nature
1996
;
384
:
577
–81.
23
Daniels MA, Jameson SC. Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.
J Exp Med
2000
;
191
:
335
–46.
24
Purbhoo MA, Boulter JM, Price DA, et al. The human CD8 coreceptor effects cytotoxic T cell activation and antigen sensitivity primarily by mediating complete phosphorylation of the T cell receptor ζ chain.
J Biol Chem
2001
;
276
:
32786
–92.
25
Buslepp J, Wang H, Biddison WE, Appella E, Collins EJ. A correlation between TCR Vα docking on MHC and CD8 dependence: implications for T cell selection.
Immunity
2003
;
19
:
595
–606.
26
Pittet MJ, Valmori D, Dunbar PR, et al. High frequencies if naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLS)-A2 individuals.
J Exp Med
1999
;
190
:
705
–15.
27
Yee C, Savage PA, Lee PP, Davis MM, Greenberg PD. Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers.
J Immunol
1999
;
162
:
2227
–34.
28
Busch DH, Pamer EG. T cell affinity maturation by selective expansion during infection.
J Exp Med
1999
;
189
:
701
–10.
29
Lyons GE, Roszkowski JJ, Mann S, Yee C, Kast WM, Nishimura MI. TCR tetramer binding and the lack there of does not necessitate antigen reactivity in TCR transduced T cells.
Cancer Immunol Immunother
2006
;
55
:
1142
–50.
30
Callender GG, Rosen HR, Roszkowski JJ, et al. Identification of a CD8-independent hepatitis C virus-specific T cell receptor that does not require CD8 for target cell recognition.
Hepatology
2006
;
43
:
973
–81.
31
Cormier JN, Panelli MC, Hackett JA, et al. Natural variation of the expression of HLA and endogenous antigen modulates CTL recognition in an in vitro melanoma model.
Int J Cancer
1999
;
80
:
781
–90.
32
Langerman A, Callender GG, Nishimura MI. Engineering bifunctional T cells as a treatment for immunotherapy resistant tumors.
J Transl Med
2004
;
2
:
42
.
33
Abraham RT, Weiss A. Jurkat T cells and development of the T-cell receptor signalling paradigm.
Nat Rev Immunol
2004
;
4
:
301
–8.
34
Alexander-Miller MA, Leggatt GR, Sarin A, Berzofsky JA. Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL.
J Exp Med
1996
;
184
:
485
–92.
35
Zeh HJ, Perry-Lalley D, Dudley ME, Rosenberg SA, Yang JC. High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy.
J Immunol
1999
;
162
:
989
–94.
36
Witte T, Spoerl R, Chang HC. The CD8β ectodomain contributes to the augmented coreceptor function of CD8αβ heterodimers relative to CD8αα homodimers.
Cell Immunol
1999
;
191
:
90
–6.
37
Bosselut R, Kubo S, Guinter T, et al. Role of CD8β domains in CD8 coreceptor function: importance for MHC I binding, signaling, and positive selection of CD8+ T cells in the thymus.
Immunity
2000
;
12
:
409
–18.
38
Leishman AJ, Naidenko OV, Attinger A. T cell responses modulated through interaction between CD8αα and the nonclassical MHC class I molecule, TL.
Science
2001
;
294
:
1936
–9.
39
Kuball J, Schmitz FW, Voss RH, et al. Cooperation of human tumor-reactive CD4+ and CD8+ T cells after redirection of their specificity by a high-affinity p53A2.1-specific TCR.
Immunity
2005
;
22
:
117
–29.
40
Tsuji T, Yasukawa M, Matsuzaki J, et al. Generation of tumor-specific, HLA class I-restricted human Th1 and Tc1 cells by cell engineering with tumor peptide-specific T-cell receptor genes.
Blood
2005
;
106
:
470
–6.
41
Pittet MJ, Rubio-Godoy V, Bioley G, et al. α3 Domain mutants of peptide/MHC class I multimers allow the selective isolation of high avidity tumor-reactive CD8 T cells.
J Immunol
2003
;
171
:
1844
–9.
42
Dutoit V, Rubio-Godoy V, Doucey MA, et al, Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR.
J Immunol
2002
;
168
:
1167
–71.
43
Choi EM, Chen JL, Wooldridge L, et al. High avidity antigen-specific CTL identified by CD8-independent tetramer staining.
J Immunol
2003
;
171
:
5116
–23.
44
Gao GF, Tormo J, Gerth UC, et al. Crystal structure of the complex between human CD8α(α) and HLA-A2.
Nature
1997
;
387
:
630
–4.