Influence of Human CD8 on Antigen Recognition by T-Cell Receptor–Transduced Cells

Research Article

Gretchen E. Lyons, Tamson Moore, Natasha Brasic, Mingli Li, Jeffrey J. Roszkowski, and Michael I. Nishimura

Department of Surgery, The University of Chicago, Chicago, Illinois

Abstract of CD8 binds the Src kinase, lck (10, 11). CD8a recruits signaling The CD8 coreceptor on T cells has two functions. Namely, components to the cytoplasmic side of the TCR/pMHC interaction CD8 acts to stabilize the binding of the T-cell receptor (TCR) (12) and provides the majority of the binding energy in the CD8 h to the peptide-MHC complex while localizing p56lck (lck)to heterodimer (13). The tail of the CD8 contains 19 residues and h the TCR/CD3 complex to facilitate early signaling events. plays a role in positive selection; however, the importance of CD8 Although both functions may be critical for efficient for the activation of T cells is not clearly defined. It has been shown aa ah activation of a CTL, little is known about how the structural that the globular heads of murine CD8 and CD8 bind to MHC versus signaling roles of CD8, together with the relative with similar affinity, and cell-to-cell adhesion assays have shown aa ah strength of the TCR, influences T-cell function. We have that CD8 and CD8 mediate adherence to pMHC equally (14, ah addressed these issues by introducing full-length and 15). Despite these data, heterodimeric CD8 has consistently aa truncated versions of the CD8A and CD8B chains into been shown to be a better coreceptor than homodimeric CD8 À CD8 Jurkat cell clones expressing cloned TCRs with known and correlates with enhanced sensitivity to peptide antigen (16). h antigen specificity and relative affinities. Using a combina- This may be due to the fact that CD8 is palmitoylated at the tion of antigen recognition and tetramer-binding assays, we cytoplasmic tail and has been shown to induce higher lck find that the intracellular lck-binding domain of CD8 is activation in lipid rafts (17). Other studies have shown that the critical for enhanced T-cell activation regardless of the glycosylation of CD8h differs between immature and mature relative strength of the TCR. In contrast, the extracellular thymocytes and that the extent of glycosylation affects its binding domain of CD8 seems to be critical for TCRs with lower to MHC class I (18, 19). Together, these data suggests a distinct role affinity but not those with higher affinity. Based on our for CD8h, but there is considerable controversy about the critical results, we conclude that there are different requirements for domains of CD8h and by what mechanism CD8h affects coreceptor CD8 to enhance T-cell function depending on the strength of function (20). its TCR. (Cancer Res 2006; 66(23): 11455-61) Studies to determine whether CD8 functions primarily to facilitate TCR binding and stabilization to pMHC, to enhance Introduction signal transduction, or a combination both, have yielded conflicting results. TCR affinity has been shown to play a significant role in The T-cell coreceptor CD8 is known to bind class I molecules determining the sensitivity of a TCR to a peptide antigen. It has directly and to be critical for development of CD8+ T cells (1, 2). It been shown that some TCRs bind pMHC despite the presence of is generally accepted that CD8 binding to MHC helps stabilize the anti-CD8 antibodies or mutations in the CD8-binding site (21), T-cell receptor (TCR)/peptide MHC (pMHC) complex and localizes whereas other TCRs require CD8 stabilization to allow sufficient the CD8-associated protein tyrosine kinase lck to the CD3complex, time for TCR/pMHC interactions (22, 23). Other studies suggest which aids in initiating the TCR signaling cascade by phosphor- that although CD8 engagement is not necessary for pMHC binding ylating the ~ chain of the TCR-associated CD3complex (1, 3–5). to the TCR, full activation of the T cell does not occur in the However, the influence of CD8 on the function of T cells expressing absence of CD8 ligation (24). A possible explanation for the high- versus low-affinity TCRs is poorly understood. We and others discrepancies between various experimental systems is that CD8 have speculated that CD8 expression is required only for those coreceptor dependency may vary with the affinity of the TCR for its TCRs with lower affinity, whereas CD8 expression is not required pMHC ligand (16, 25). Strong agonist-peptide MHC complexes for T cells expressing higher-affinity TCRs (6–8). Based on these activate T cells without the involvement of CD8-pMHC interaction, studies, the contribution of CD8 to TCR/pMHC stability seems to whereas for weak ones, this interaction is necessary (26). Analysis be more important than colocalizing lck to the CD3complex for using pMHC tetramers have found a correlation between tetramer T-cell activation. CD8 is encoded by two distinct genes, CD8a and CD8b, and is binding, which is generally accepted as a measure of TCR affinity, expressed on the surface of T cells primarily as a CD8ah and the amount of antigen required to elicit a functional response heterodimer (9). The Ig domain of CD8 binds to the conserved (27–29). However, it has also been shown that some CTL clones a3domain of MHC class I molecules, whereas the cytoplasmic tail 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 Requests for reprints: Michael I. Nishimura, Department of Surgery, Medical University of South Carolina, Hollings Cancer Center, 86 Jonathon Lucas Street, Suite that exhibit CD8-independent recognition of tumor cells (29, 30). 512, P.O. Box 250613, Charleston, SC 29425. Phone: 843-792-7789; Fax: 843-792-2556; Thus, factors other than TCR affinity can be important in E-mail: [email protected]. I2006 American Association for Cancer Research. determining the sensitivity to peptide antigen, and the CD8 doi:10.1158/0008-5472.CAN-06-2379 coreceptor may play an important role in both functions. www.aacrjournals.org 11455 Cancer Res 2006; 66: (23). December 1, 2006

To better define how the cytoplasmic and extracellular portions 72jC (35 cycles), followed by 5 minutes at 72jC (one cycle). This allowed for of CD8 contribute to antigen recognition by T cells, we created full- the amplification of either full-length CD8a and CDh or truncated CD8a h a¶ h¶ length and truncated versions of these molecules, which lack the and CD ( or ). The resulting PCR products were separated on 1% lck-binding domain and introduced them into Jurkat clones agarose gels containing ethidium bromide (Continental Lab Products) and were visualized under UV light. The products of each PCR reaction were expressing high- or low-affinity–cloned TCRs reactive with known ligated into the pCR 2.1 TA cloning vector (Invitrogen) and transformed melanoma antigens. Here, we show that the stabilization and into Escherichia coli DH5a-competent cells (Invitrogen). Bacterial clones signaling properties of the CD8 coreceptor are separate entities. were screened by PCR using the specific CD8a and CD8h primers to Namely, although the intracellular signaling domain significantly identify clones containing inserts of the predicted size. DNA was isolated enhances sensitivity to peptide antigen as well as tumor cell from those clones and their inserts were sequenced by cycle sequencing recognition, it does not contribute to tetramer binding. Further- using BigDye Terminator Cycle Sequencing kits (Applied Biosystems, more, depending on the relative affinity of the TCR, TCR/pMHC Foster City, CA) and analyzed on an ABI Prism 310 Genetic Analyzer stabilization afforded by the CD8 coreceptor can contribute to (Applied Biosystems) to ensure there were no PCR errors in the sequence. T-cell function. Retroviral vector construction. The SAMEN cytomegalovirus (CMV)/SRa retroviral vector was designed specifically for introducing TCR and accessory molecules into alternate T cells. Construction of TIL Materials and Methods 1383I and TIL 5 TCR retroviral constructs has been described previously Cells. Cell lines were purchased from American Type Culture Collection (7, 8). Briefly, the 5¶ long terminal repeat (LTR) in the SAMEN SRa (Manassas, VA) unless otherwise noted. Jurkat cells are a CD8-negative backbone was replaced with a hybrid LTR consisting of the human CMV À À human T-cell lymphoma and T2 cells are a Tap-1 / human HLA-A*0201 B/ enhancer and promoter fused to the Moloney murine leukemia virus T lymphoma. Melanoma cell lines, mel 1300 and mel 624, are HLA-A2 5¶ LTR. This modification permits production of retroviral supernatants positive, whereas mel 888 is HLA-A2 negative (31). All of these cells were by transiently transfecting 293GP cells (8). Other key elements of the maintained in human complete medium, which consisted of RPMI 1640 SAMEN CMV/SRa vector include an internal SRa promoter to permit (Mediatech, Herndon, VA) supplemented with 10% FCS (Gemini Bio- the expression of multiple genes, unique SalIandXhoI restriction sites Products, West Sacramento, CA) and 100 units/mL penicillin (Mediatech), for ease of inserting TCR chains, and an internal ribosome entry site/ r 100 Ag/mL streptomycin (Mediatech), and 2.92 mg/mL L-glutamine neo cassette for G418 selection. A rapid ligation strategy was used to (Mediatech). 293GP retroviral producer cells were maintained in DMEM subclone the CD8a and CD8b chain genes into SAMEN CMV SRa. CD8a (Mediatech) supplemented as above. The original TIL 5 and TIL 1383I lines and CD8a¶ genes were excised from pCR2.1 with XhoI and ligated into were established from surgical specimens obtained from melanoma patients the SalI restriction site in SAMEN CMV/SRa using a mixture of T4 DNA undergoing immunotherapy in the Surgical Branch, National Cancer ligase and SalI restriction endonuclease. The resulting SalI/XhoIhybrid Institute. T-cell clones were grown in X-Vivo 15 (Cambrex, Walkersville, sites are resistant to digestion by SalIandXhoI. Ligation reactions were MD) medium supplemented with 10% heat-inactivated human AB serum redigested with SalI, resulting in linearization of plasmids not (Valley Biomedical, Winchester, VA), 100 units/mL penicillin (Invitrogen, containing the CD8a chain inserts, allowing for enrichment of Carlsbad, CA), 100 Ag/mL streptomycin (Invitrogen), 2.92 mg/mL recombinant clones. The ligation reactions were cloned into E. coli L-glutamine (Invitrogen), and 6,000 IU/mL recombinant human interleukin DH5a-competent cells (Invitrogen), and bacterial clones were screened 2 (IL-2; Chiron, Emeryville, CA). Because the parental TIL 5 clone is no longer by PCR using primers that flanked the cloning sites in SAMEN CMV/ a available for analysis, another MART-127-35–reactive T-cell clone was used SR . The DNA sequence of clones containing inserts was determined as a positive control for HLA-A*0201 MART-127-35 tetramer staining. using BigDye Terminator Cycle Sequencing kits and analyzed using an Reagents and antibodies. The HLA-A*0201–binding peptides MART- ABI Prism 310 Genetic Analyzer (Perkin-Elmer/ABI, Foster City, CA) to

127-35 (AAGIGILTV) and tyrosinase368-376 (YMDGTMSQV) were synthesized ensure that CD8 gene inserts were in the proper orientation. This at Macromolecular Resources (Fort Collins, CO) and purified by reversed- method was repeated to insert the CD8h chains into the retroviral phase high-performance liquid chromatography (HPLC). Purity was vector by ligating a SalIfragmentcontainingtheh-chain into the XhoI assessed by analytic HPLC and was determined to be >99%. Anti-human site of SAMEN CMV/SRa to create two CD8 retroviruses, CD8ah and CD4-FITC and anti-human CD8a-FITC were purchased from BD Bio- CD8a¶h¶ (Fig. 1). sciences (San Diego, CA). Anti-human CD8 h-phycoerythrin was purchased Generation of retroviral supernatants. One hundred millimeter tissue from Immunotech (Marseilles, France). Anti-human Vh12-FITC was culture plates were coated with 0.2% type B bovine skin gelatin (Sigma) in purchased from Pierce Biotechnology (Rockford, IL). HLA-A*0201 tetramers HBSS for 15 minutes at room temperature. 293GP cells were seeded onto labeled with phycoerythrin and containing the MART-127-35 and tyrosi- coated plates at sufficient density to provide 70% confluency after 24 hours f Â 6 nase368-376 peptide were obtained from Beckman Coulter (Fullerton, CA). ( 3 10 ). Monolayers were rinsed thrice with PBS and transiently Gene identification, cloning, and analysis. The identification and cotransfected using LipofectAMINE and PLUS reagents (Life Technologies), cloning of TCR a and h chains from the TIL 5 and TIL 1383I have been 3 Ag of the retroviral plasmid containing the CD8 genes, and 3 Ag vesicular described elsewhere (6, 8). The cloning of the CD8a and CD8h chains stomatitis virus envelope gene in 6.0 mL serum-free DMEM. Following a was done by reverse transcription-PCR using CD8a- and CD8h-specific 3-hour incubation, 10 mL DMEM with 20% FCS was added to the flasks, and primers for the full-length and truncated versions of each chain. Total cells were incubated at 37jC. Medium was discarded after 24 hours and RNA was isolated from 5 Â 106 T cells using RNAeasy kits (Qiagen, replaced with 10 mL human complete medium. Retroviral supernatants Valencia, CA) according to the instructions of the manufacturer. First- were collected after 24 hours, replaced with medium, and collected again strand cDNA was prepared from 1 Ag total RNA using Superscript II after an additional 24 hours. Retroviral supernatants were either used À j reverse transciptase (Invitrogen) and oligo(dT)12-18 (Invitrogen). Ten immediately or frozen at 70 C for later use. nanograms of cDNA were PCR amplified in a 50 AL reaction consisting of Retroviral transduction. Fresh retroviral supernatants were supple- Â A A 1 PCR buffer (Invitrogen), 1.5 mmol/L MgCl2 (Invitrogen), 200 mol/L mented with 8 g/mL polybrene (Sigma) and filter sterilized. Jurkat cells deoxynucleotide triphosphate (Invitrogen), 400 nmol/L CD8a-orCD8h- were suspended at 1 Â 106/mL in retroviral supernatant. One milliliter of specific forward primer, 400 nmol/L CD8a or CD8h chain intracellular cells per supernatant was added to each well of 24-well tissue culture region specific or CD8a or CD8h transmembrane reverse primers, and 2.5 plate, and the plates were centrifuged at 1,000 Â g for 90 minutes at 32jC. units Taq DNA polymerase (Continental Lab Products, San Diego, CA). Plates were returned to the incubator and, after 4 hours, 1 mL fresh RPMI PCR amplification was done using a MJ Research (Watertown, MA) thermo- was added to each well. Transduced cells were incubated overnight, and cycler under the following conditions: 5 minutes at 92jC (one cycle) this procedure was repeated the next day with fresh supernatant as followed by 30 seconds at 92jC, 30 seconds at 58jC, and 1 minute at described above.

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Results 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 (6–8, 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 Figure 1. Structure of the wild-type and truncated CD8. Retroviral vectors antigen did not always correlate with its affinity for the tetramer. encoding CD8 were used to transduce Jurkat cells to express the wild-type or This underscores the importance of defining the contributions of truncated CD8ah heterodimers. A, the CD8 retroviruses were created by CD8 on the TCR affinity for tetramer and avidity for antigen. inserting the CD8a and CD8b genes into the SAMEN CMV/SRa retroviral vector. The vector is composed of a Moloney murine leukemia virus backbone. The 5¶ To better define the role of CD8 in TCR/pMHC interactions and LTR has been modified by replacing the R and U5 segments with a CMV IE tumor cell recognition, we transduced Jurkat clones expressing the promoter enhancer to promote high-level transient expression in 293GP producer cells. Other key elements include the c+ packaging signal, splice donor TIL 5 (clone 22) or TIL 1383I (clone C4) TCRs with retroviral and splice acceptor, an internal SRa promoter to allow expression of a second vectors encoding wild-type CD8ah or CD8a¶h¶ (Fig. 1). Jurkat cells gene, CD8h and CD8a chains inserted into unique SalI and XhoI restriction were used as a readout for T-cell activation and function in these 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 studies because they are easily gene modified (6, 8, 30) and their molecules used in these studies. CD8ah depicts the wild-type genes of the signaling properties have been well characterized (33). As shown in heterodimer, including the lck-binding domain on the intracellular a chain. Fig. 2, the resulting Jurkat cells expressed high levels of CD8ah or CD8a¶h¶ depicts the truncated copies of both the a and h heterodimer. CD8a¶h¶ heterodimers. CD8a¶h¶ lack cytoplasmic tails and therefore cannot colocalize lck to the CD3complex; however, its Antigen recognition assay. Retrovirally transduced Jurkat cells were intact extracellular a domain is still capable of binding to MHC I. tested for reactivity to tumor antigens in cytokine release assays. Tumor The expression of CD8ah or CD8a¶h¶ on TIL 1383I TCR Jurkat cells A cells or T2 cells preincubated for 2 hours with 10 g/mL peptides were had little effect on TCR expression because Vh12 levels were washed twice with PBS and then added to effector cells at a 1:1 ratio in similar in the untransduced, CD8ah-transduced, or CD8a¶h¶- a total volume of 200 AL human complete medium per well of a 96-well, U-bottomed tissue culture plate. Actual numbers were 2 Â 105 cells per well. transduced Jurkat clone C4 cells (Fig. 3). Similar analysis could not be done on TIL 5 TCR Jurkat cells because there are no Cocultures were incubated at 37jC in a humidified CO2 incubator for 24 hours. Supernatants were harvested, and the amount of human IL-2 antibodies available that are capable of staining the TIL 5 TCR (6). (R&D Systems, Minneapolis, MN) released by Jurkat cells was measured by We have found that the introduction of the CD8ah molecule into ELISA (R&D Systems). TIL 1383I TCR Jurkat cells resulted in tetramer binding, whereas

Figure 2. Expression of CD8ah and CD8a¶h¶ 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, CD8ah-containing, or CD8a¶h¶-containing SAMEN CMV/SRa retroviral vector. Stable transductants were stained for expression of either CD8a-FITC or CD8 h-phycoerythrin and fluorescence was quantified by flow cytometry. Histograms, relative log fluorescence of 104 viable cells.

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mel 1300 cells but not mel 624 when expressing CD8ah heterodimers. The most likely explanation for the differential recognition of the melanomas by CD8ah 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 CD8ah 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). 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 CD8ah (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 Figure 3. Relationship between CD8 and TCR expression on TIL 1383I TCR of CD8 in antigen recognition by TIL 5 and TIL 1383I TCR Jurkat cells. The levels of CD8 and Vh12expression were measured on TIL Jurkat cells, cells transduced to express the a¶h¶ form of CD8, 1383I TCR Jurkat cells to ensure that the retroviral transduction did not which lacks the lck-binding domain, were assayed for recogni- significantly alter the expression of the TIL 1383I TCR. Untransduced clone C4 cells and clone C4 cells transduced with the empty, CD8ah-containing, or tion of peptide-loaded T2 cells and melanoma cells. As shown in CD8a¶h¶-containing SAMEN CMV/SRa retroviral vector were stained for Figs. 4B and 5A,theexpressionofCD8a¶h¶ had different effects a h expression of CD8 (anti-CD8 -phycoerythrin) or the TIL 1383I TCR (anti-V 12- on IL-2 secretion depending on the TCR expressed. When FITC). Fluorescence staining was quantified by flow cytometry. Histograms, relative log fluorescence of 104 viable cells. CD8a¶h¶ 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 cells transduced with the empty vector failed to bind tetramers expression of CD8a¶h¶ stabilized the TCR/pMHC complex as (Fig. 4A). CD8a¶h¶ expressing TIL 1383I TCR Jurkat cells could also illustrated by the ability of these cells to bind tetramers bind tetramers, indicating that the cytoplasmic tails had no effect (Fig. 4A). Therefore, it is the signaling function, and not the on the ability of CD8 to stabilize the TCR/pMHC complex (Fig. 4A). MHC-binding function of CD8, that influences antigen recogni- Although the expression of CD8 facilitated tetramer binding to TIL tion by TIL 1383I TCR–transduced Jurkat cells. In contrast, 1383I TCR-expressing cells, TIL 5 TCR Jurkat cells with or without expression of CD8a¶h¶ in TIL 5 TCR Jurkat cells led to enhanced CD8 failed to bind tetramers (data not shown). These results IL-2 release relative to cells transduced with the empty vector indicate that CD8 can facilitate tetramer binding to some, but not when stimulated with peptide-loaded T2 cells (Fig. 5A). Like all, TCRs. cells expressing the wild-type CD8 molecule, cells expressing Influence of CD8 on antigen recognition by TCR-trans- CD8a¶h¶ could recognize mel 1300, whereas cells transduced duced Jurkat clones. In addition to TCR/pMHC stabilization, with the empty vector could not. The amount of IL-2 released by we analyzed the effect of the structural domains of CD8 on CD8a¶h¶ expressing TIL 5 TCR Jurkat cells was less than CD8ah antigen recognition by TCR-transduced Jurkat cells. To assess expressing TIL 5 TCR Jurkat cells, indicating that colocalization the capacity of the different forms of CD8 to affect antigen of lck to the CD3complex by CD8 was also important for tumor recognition, we did cytokine release assays using our panel of cell recognition by TIL 5 TCR-expressing cells. Based on these TCR-transduced Jurkat cells. Each Jurkat clone transduced with results, we conclude that stabilizing the TCR/pMHC complex by the empty vector, CD8ah,orCD8a¶h¶ was cocultured with T2 CD8ah heterodimers can increase the efficiency of antigen cells loaded with the MART-127-35 or tyrosinase368-376 peptide, recognition by TCR-transduced cells, and, depending on the HLA-A2–positive tumor cell lines (mel 1300 and mel 624), or an strength of the TCR, CD8 expression is essential for allowing HLA-A2–negative tumor cell line (mel 888). Responder and tetramer binding and for tumor cell recognition to occur. stimulator cells were cocultured overnight. The next day, Influence of CD8 on the functional avidity of Jurkat supernatants were removed and the amount of IL-2 was clones. It has been argued that tumor cell recognition is related measured by ELISA. to the functional avidity or sensitivity of a T-cell to antigen The addition of CD8ah consistently showed the greatest stimulation (34, 35). To determine how the structural versus increase in IL-2 production when compared with empty vector signaling features of CD8 influence functional avidity, we controls for both TIL 5 (Fig. 5A) and TIL 1383I (Fig. 4B) TCR Jurkat cocultured our Jurkat clones transduced with the empty vector, cells. However, the TIL 5 TCR Jurkat cells, which previously only CD8ah,orCD8a¶h¶ with T2 cells loaded with differing recognized peptide-loaded targets (6), were now able to recognize concentration of either the MART-127-35 or the tyrosinase368-376

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. CD8 and Antigen Recognition peptides. Responder and stimulator cells were cocultured well-characterized TCRs. This approach allowed us to separate the overnight, supernatants were removed the next day, and the structural and signaling features of CD8 to determine which is amount of IL-2 was measured by ELISA. The expression of more important for T-cell function. CD8ah increased the sensitivity of both TIL 5 (Fig. 5B)andTIL Results obtained in this study challenge the hypothesis that 1383I (Fig. 4C) TCR Jurkat cells to antigen stimulation compared increased TCR affinity through the addition of CD8 leads to with the empty vector controls (Figs. 4C and 5B). As we have enhanced T-cell function. Based on the analysis of two different observed with tumor cell recognition, the influence of CD8a¶h¶ TCRs, we find that it is the affinity of the TCR for their pMHC on functional avidity depended on which TCR was expressed. ligand that dictates its need for CD8. Furthermore, the Expression of CD8a¶h¶ on TIL 1383I TCR Jurkat cells had little differences in the CD8a/CD8h ratios between TIL 5 and TIL effect on their sensitivity to antigen stimulation (Fig. 4C). These 1383I TCR–transduced Jurkat cells or the requirement of CD8h cells were only slightly more sensitive to antigen than cells do not effect on our results. TIL 5 and TIL 1383I Jurkat cells transduced with the empty vector, which required f10-fold expressing CD8 are similar to TCR-transduced T-cell clones in more peptide to activate the cells than CD8ah TIL 1383I TCR Jurkat cells. In contrast, expression of CD8a¶h¶ 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 CD8a¶h¶ TIL 5 TCR Jurkat cells to antigen stimulation was less than CD8ah 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.

Discussion Many studies have been aimed at determining the relationships between T-cell function, TCR affinity, and CD8 dependence (36–38). 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 (41–43). 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

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, CD8ah-containing, or CD8a¶h¶-containing SAMEN CMV/SRa 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, CD8ah-containing, or CD8a¶h¶-containing SAMEN CMV/SRa retroviral vector were cocultured overnight with peptide- loaded T2cells ( 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-A2À). 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 (.), CD8ah-containing (E), or CD8a¶h¶-containing (!) SAMEN CMV/SRa retroviral vector were cocultured overnight with T2cells 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-2release (<32pg/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. www.aacrjournals.org 11459 Cancer Res 2006; 66: (23). December 1, 2006

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 CD8a¶h¶. 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 CD8ah versus CD8a¶h¶ coreceptors, we were able to determine to what extent colocalizing lck to the CD3complex influenced T-cell activation. Here again, the results obtained were dependent on the TCR. In all cases, the ability of CD8ah to colocalize lck to the CD3complex 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 CD8ah, but not CD8a¶h¶, 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 CD3complex and, thus, it is signaling rather than the stabilization of the TCR/p/MHC Figure 5. Influence of CD8 expression on antigen recognition by TIL 5 TCR complex that is responsible for the increase in T-cell activity Jurkat cells. The influence of CD8 expression on Jurkat cells expressing the TIL afforded by CD8. This conclusion is supported by structural studies 5TCR was determined. A, the influence of CD8 expression on antigen recognition was determined. Clone 22 cells transduced with the empty, that indicate that the binding of one molecule is unlikely to alter CD8ah-containing, or CD8a¶h¶-containing SAMEN CMV/SRa retroviral vector the affinity for the other (44). We should make it clear that we do were cocultured overnight with peptide-loaded T2cells ( striped columns)or not entirely discount the effect of the contribution of the melanoma cell lines (filled columns). Peptides used were MART-127-35 and + tyrosinase368-376. Melanoma cells used were mel 1300 (HLA-A2 ), mel 624 extracellular domain in enhancing the TCR/pMHC interaction. (HLA-A2+), and mel 888 (HLA-A2À). The antigen recognition was assessed by Clearly, the simultaneous binding of the TCR and CD8 by pMHC the release of IL-2. The amount of cytokine released was measured by ELISA. Columns, average of triplicate wells. Representative of three replicate allows potential for CD8-induced stabilization of the TCR/pMHC experiments. B, the influence of CD8 expression on the functional avidity or interaction. Despite the low affinity of the pMHC/CD8 interaction, sensitivity of TIL 5 TCR Jurkat cells to antigen stimulation was determined. any such stabilization could be of extreme biological importance. Clone 22 cells transduced with the empty (.), CD8ah-containing (E), or CD8a¶h¶-containing (!) SAMEN CMV/SRa retroviral vector were cocultured This is clearly shown by the need for CD8 in normal T cells overnight with T2cells loaded with five different concentrations of MART-1 27-35 expressing the TIL 5 and other lower-affinity TCRs (8, 30, 39, 40). peptide. The antigen recognition was assessed by the release of IL-2. The When considering strategies to enhance the function of normal amount of cytokine released was measured by ELISA. There was no IL-2release + + (<32pg/mL, which was below the lowest value on the standard curve) against an CD8 T cells or TCR-transduced CD8 T cells for increased irrelevant tyrosinase368-376 control peptide. Points, average of triplicate wells. therapeutic efficacy, altering the stability of the TCR/pMHC Representative of three independent experiments. 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 their ability to bind tetramers and recognize antigen. The TIL 5 the affinity of the TCR for its pMHC ligand. Strategies to enhance TCR, which has lower affinity, supports the aforementioned T-cell function by altering their signaling pathways using cytokines hypothesis because TIL 5 TCR Jurkat cells expressing CD8a¶h¶ and reversing inhibitory signals are currently being explored. 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 Acknowledgments CD8a¶h¶ to recognize mel 1300 cells, whereas cells transduced Received 6/29/2006; revised 8/31/2006; accepted 9/15/2006. with the empty vector cannot. In contrast, the higher-affinity Grant support: NIH grants CA90873, CA111040, and CA102280. The costs of publication of this article were defrayed in part by the payment of page TIL 1383I TCR does not support this hypothesis. Despite the fact charges. This article must therefore be hereby marked advertisement in accordance that CD8a¶h¶ expression allows tetramers to bind to TIL 1383I with 18 U.S.C. Section 1734 solely to indicate this fact.

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