Kinase Activation and CD8 T Cell Development

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1 This information is current as of February 23, The Cytoplasmic Domain of CD8β Regulates Lck Kinase Activation and CD8 T Cell Development Hanna Yoko Irie, Mimi S. Mong, Andrea Itano, M. E. Casey Crooks, Dan R. Littman, Steven J. Burakoff and Ellen Robey J Immunol 1998; 161: ; ; References Subscriptions Permissions Alerts This article cites 34 articles, 13 of which you can access for free at: Information about subscribing to The Journal of Immunology is online at: Submit copyright permission requests at: Receive free -alerts when new articles cite this article. Sign up at: The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD Copyright 1998 by The American Association of Immunologists All rights reserved. Print ISSN: Online ISSN:

2 The Cytoplasmic Domain of CD8 Regulates Lck Kinase Activation and CD8 T Cell Development 1 Hanna Yoko Irie, 2 * Mimi S. Mong, 2 Andrea Itano, M. E. Casey Crooks, Dan R. Littman, Steven J. Burakoff, 3 * and Ellen Robey Previous studies have shown that CD8 plays a role in both enhancing CD8 -associated Lck kinase activity and promoting the development of CD8-lineage T cells. To examine the role of this enhancement in the maturation of CD8-lineage cells, we assessed CD8 -associated Lck kinase activity in both T cell hybridomas and thymocytes of mice expressing CD8 mutations known to impair CD8 T cell development. Lack of CD8 expression or expression of a cytoplasmic domain-deleted CD8 resulted in a severalfold reduction in CD8 -associated Lck kinase activity compared with that observed with cells expressing wild-type CD8 chain. This analysis indicated a critical role for the cytoplasmic domain of CD8 in the regulation of CD8 -associated Lck activity. Decreased CD8 -associated Lck activity observed with the various CD8 mutations also correlated with diminished in vivo cellular tyrosine phosphorylation. In addition, analysis of CD8 mutant mice (CD8 / or cytoplasmic domain-deleted CD8 transgenic) indicated that the degree of reduction in CD8 -associated Lck activity associated with each mutation correlated with the severity of developmental impairment. These results support the importance of CD8 -mediated enhancement of CD8 associated Lck kinase activity in the differentiation of CD8 single-positive thymocytes. The Journal of Immunology, 1998, 161: CD8 is expressed predominantly on the surface of class I MHC-restricted T cells, either as a homodimer of two -chains or as a heterodimer of an - and a -chain. While most thymocytes and peripheral T cells express the heterodimeric form of CD8, specific subpopulations of NK cells and intestinal T cells exclusively express CD8. The CD8 molecule is a 30-kDa glycoprotein that is expressed on the cell surface only in association with an -chain (1 7). Possible roles for the CD8 chain in T cell function include influencing the avidity and/or specificity of the CD8/MHC/TCR interaction (8, 9) and enhancing IL-2 production in response to stimulator cells (10). Recent studies also suggested that the ability of T cells to recognize altered peptide ligands as full or partial agonists depended upon the expression of CD8 ; coexpression of CD8 and CD8 resulted in a pattern of T cell activation, as indicated by calcium mobilization, characteristic of full agonists (11). Our previous studies demonstrated that the CD8 chain enhances the activity of Lck tyrosine kinase associated with the CD8 chain, and that this enhancement correlates with greater association of Lck with CD8 *Department of Pediatric Oncology, Dana-Farber Cancer Institute, and Department of Pediatrics, Harvard Medical School, Boston, MA 02115; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Department of Microbiology and Immunology, University of California, San Francisco, CA 94143; and Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY Received for publication December 24, Accepted for publication March 3, 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 This work was supported by National Research Service Award Training Grant GMO (to H.Y.I.), National Institutes of Health Grant AI17258 (to S.J.B.), and National Institutes of Health Grant RO1AI32985 (to E.R.). 2 These authors contributed equally to this work. (12). The region of CD8 that is responsible for this enhancement, however, has not yet been identified. The importance of the CD8 chain in thymic development was also suggested by several studies. Several groups generated mice bearing a homozygous disruption of the CD8 gene, and it was observed that the thymic development of mature CD8 T cells was significantly impaired, resulting in an 80% reduction in the number of mature peripheral CD8 T cells. The absence of CD8 chain expression also resulted in impaired positive selection of thymocytes expressing a known transgenic TCR (13 15). Although the mechanisms by which CD8 regulates these developmental processes have not yet been elucidated, the cytoplasmic domain appears to play a crucial role. The cytoplasmic domain of CD8 is highly conserved across species and consists of only 19 amino acids (6). Unlike the cytoplasmic domain of CD8, which contains a motif for association with Lck (16), there are no known protein binding motifs in the cytoplasmic region of CD8 (17). Nevertheless, expression of a cytoplasmic tail-deleted mutant CD8 chain (CD8 (TL)) 4 as a transgenic construct in wild-type mice inhibited the development of CD8 T cells by twofold. This mutant appeared to function in a dominant negative manner, competing and interfering with the function of the endogenous, wildtype CD8 chain (18). While these studies indicated a role for CD8 and its cytoplasmic domain in the development of CD8 T cells, it is not clear whether the role of CD8 in development is related to its role in enhancing CD8-associated Lck activity. To assess potential mechanisms that may account for the impaired development of CD8 T cells in CD8 / or CD8 (TL) mice, we examined in this present study the effect of either lack of CD8 expression or deletion of the CD8 cytoplasmic domain on CD8 -associated Lck kinase activity. In our analysis of both hybridomas and thymocytes, both types of CD8 mutations resulted in reduced CD8 -associated Lck kinase activity. As deletion of the 3 Address correspondence and reprint requests to Dr. Steven J. Burakoff, DANA 1640, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA address: steven burakoff@macmailgw.dfci.harvard.edu 4 Abbreviations used in this paper: CD8 (TL), cytoplasmic tail-deleted CD8 ; CD8 (wt), wild-type CD8 ; RAMG, rabbit anti-mouse IgG; PVDF, polyvinylidene difluoride; ECL, enhanced chemiluminescence; PE, phycoerythrin. Copyright 1998 by The American Association of Immunologists /98/$02.00

3 184 CD8 CYTOPLASMIC TAIL REGULATES Lck AND CD8 T CELL DEVELOPMENT cytoplasmic domain nearly completely abrogated activation of CD8 -associated Lck mediated by wild-type CD8, our data suggest that the 19-amino acid cytoplasmic domain of CD8 is responsible for the regulation of CD8 -associated Lck activity. Tyrosine phosphorylation of intracellular substrates, observed upon cross-linking of wild-type CD8 was also diminished in the absence of the CD8 cytoplasmic domain. Furthermore, the degree of reduction in CD8 -associated Lck activity observed with CD8 mutations correlated with the severity of impairment of CD8 T cell development. The compromised development of CD8-lineage cells in the absence of CD8 or in the presence of the tailless molecule may therefore be a consequence of the decrease in Lck kinase activation and in tyrosine phosphorylation of key intracellular substrates. Materials and Methods Constructs The human CD8.1 cdna (4) was subcloned into the EcoRI site of the expression vector pmh-neo (19), as previously described (12). A mutant CD8 cdna construct lacking 17 of the 19 residues in the cytoplasmic domain (CD8 TL) was generated by PCR using the primers: 5 -CCA CCA AGC TTG TCT CCG CCG AGC CCC CGG GGC CAG G-3 and 5 -CCC TCA CCG GTA GGT GGA CCC TAG GAT TGA GCT CGC GC-3. The mutant CD8 cdna was then subcloned into the XhoI site of pmh-neo. Cell lines and transfections BYDP, a murine T cell hybridoma expressing human CD4 and CD8 molecules (20, 21) was transfected either with the wild-type or mutant CD8 cdna constructs. Cells transfected with the pmh-neo vector alone served as controls. Cell lines that expressed comparable levels of wild-type CD8 (CD8 (wt)) or mutant CD8 (TL) heterodimer were generated. For all transfections, cells were electroporated with 10 to 20 g of DNA linearized with XmnI. Selection with 3 mg/ml G418 (Life Technologies, Gaithersburg, MD) solution was started 48 h after electroporation, and transfectants were selected for approximately 2 wk. The data presented are from representative clones. Hybridomas were analyzed with a Becton Dickinson FACScan using Abs to TCR (F23.1), CD4 (Leu 3a, Becton Dickinson, Mountain View, CA), CD8 (Leu 2a, Becton Dickinson), and CD8 (2ST8-5H7, gift from Dr. E. Reinherz, Dana-Farber Cancer Institute, Boston, MA). Stimulation, immunoprecipitations, and in vitro kinase assays Transfected hybridoma cells ( cells/ml) were incubated for 10 min on ice with the indicated Abs at the following concentrations: anti-tcr Ab (F23.1), 500 ng/ml; anti-cd4 and anti-cd8, 500 ng/ml (Becton Dickinson); and anti-cd8 Ab, 1/500 dilution of ascites. After addition of rabbit anti-mouse IgG (RAMG; 10 g/ml final concentration; Southern Biotechnology Associates, Birmingham, AL) and an additional incubation on ice for 10 min, the cells were stimulated at 37 C for 3 min, washed, and lysed as described previously (22). For T cell hybridomas, immunoprecipitations and in vitro kinase assays were performed as described previously (22). Hybridoma cells were lysed in lysis buffer containing 1% Brij-96, 50 mm Tris (ph 7.6), 150 mm NaCl, 1mMNa 3 VO 4, 10 mm NaF, leupeptin and aprotinin (10 g/ml each), and 2 mm PMSF. Lysates were incubated with 50 l of a 50% solution of protein A-Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) without further addition of Abs for 2 to 18 h at 4 C. The beads were then washed, resuspended in 50 l of kinase reaction buffer (10 mm MnCl 2,5mM HEPES, 5 mm p-nitrophenylphosphate, 10 Ci [ - 32 P]ATP, 0.1 mm Na 3 VO 4, and 10 g/ml each of aprotinin and leupeptin) and incubated at 30 C for 3 min. The proteins were resolved by 8% SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), and developed by autoradiography. Thymocytes were cultured in RPMI culture medium-rpmi 1640 supplemented with 10% FBS (Sigma, St. Louis, MO), 1% penicillin-streptomycin (Fisher, Pittsburgh, PA), 1% L-glutamine (Fisher), and 0.034% -ME for 14 h at 37 C. Cells (10 7 ) were then incubated for 15 min on ice with saturating amounts of IgM anti-murine CD4 (RL172.4) or IgM antimurine CD8 (3.155; gifts from Drs. J. Allison and D. Raulet, University of California-Berkeley). The cells were stimulated at 37 C for 3 min, washed once with HBSS (Fisher), and lysed in 750 l of ice-cold lysis buffer containing 50 mm Tris (ph 7.5), 150 mm NaCl, 1 mm Na 3 VO 4,10mM NaF, 10 mm Na 2 P 2 O 8, 1% Brij 97, 1 mm PMSF, and 10 g/ml each of pepstatin A, aprotinin, and leupeptin. After incubation on ice for 10 min, 25 l of goat anti-rat IgM (15.8 g/ml; Cappell, Durham, NC) on protein G-Sepharose beads was added to the lysates. The mixture was rocked for 2 h at 4 C, and the beads were then washed three times with ice-cold wash buffer (50 mm Tris (ph 7.5), 150 mm NaCl, and 10 mm MnCl 2 ) and resuspended in 20 l of kinase reaction buffer with 10 Ci of [ - 32 P]ATP. The kinase reaction was performed for 3 min at 30 C and was stopped by addition of 10 l of3 SDS-PAGE sample buffer. The samples were boiled for 5 min, and the proteins were resolved by 8.5% SDS-PAGE gel. The gel was soaked in a solution of 10% AcOH and 40% MeOH for 15 min, dried, and visualized by autoradiography. Anti-Lck, anti-phosphotyrosine, and anti-cd8 immunoblotting For anti-lck immunoblotting, hybridoma cells/sample were lysed in 1% Nonidet P-40 detergent; immunoprecipitated with Leu 3a, Leu 2a, or mouse IgG1 (1.25 g); resolved by 10% SDS-PAGE; transferred onto PVDF membrane; immunoblotted with anti-lck Ab directed to the C-terminus (Upstate Biotechnology, Lake Placid, NY); and developed by enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) (12). The same PVDF membrane was subsequently immunoblotted with 1 g/ml of anti-cd8 Ab directed against the carboxyl terminus (Santa Cruz Biotechnology, Santa Cruz, CA). For analysis of thymocytes, cells were lysed at cells/ml in lysis buffer and placed on ice for 10 min. Lysates were incubated for 2 h at 4 C with 50 l of protein G beads (GammaBind G Sepharose, Pharmacia, Piscataway, NJ) directly coupled to 10 mg/ml of anti-cd4 (GK1.5) or anti-cd8 ( ; gifts from Drs. D. Raulet and J. Allison, University of California, Berkeley). Beads were washed twice with lysis buffer without detergent. The samples were resolved by 10% SDS-PAGE and transferred to nitrocellulose (Hybond-ECL, Amersham). Immunoblotting was performed using ECL. Anti-Lck Ab was generated by injecting glutathione- S-transferase-Lck fusion protein, provided by Dr. Joe Bolen (Bristol-Meyers Squibb, Princeton, NJ), into rabbits, and was tested by Western blots. Blots were visualized using donkey anti-rabbit Ig horseradish peroxidase (Amersham). Immunoprecipitations with anti-phosphotyrosine Ab 4G10 (Upstate Biotechnology) were performed using lysates from stimulated hybridoma cells/sample. Lysates were incubated with 2 g of 4G10 Ab and 50 l of protein A-Sepharose beads (preincubated with RAMG) for 2 to 18 h at 4 C. The beads were washed, and the bound proteins were eluted with 10 mm p-nitrophenylphosphate as described previously (22). The proteins were resolved by SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with anti-phosphotyrosine Ab (RC20H, Transduction Laboratories, Lexington, KY). Analysis of T cell populations CD8 / and CD8 (TL) transgenic mice have been previously described (13, 18). Transgenic mice expressing CD8 (TL) with two copies of endogenous CD8 were mated with CD8 / to generate CD8 (TL) endogenous CD8 / mice. Transgenic offspring were identified by Southern blot and dot blot hybridization. For analysis by flow cytometry, thymus and lymph nodes (cervical, axillary, brachial, and mesenteric) were teased apart in cold medium M199 (Life Technologies) supplemented with 5% FBS, and the cells were filtered through nylon mesh. Staining was performed on 10 6 cells by incubating cells with 10 l of Ab on ice for 20 min. Cells were then washed twice with staining buffer containing 1 HBSS, 0.2% sodium azide, and 0.2% bovine albumin (Sigma). Data (50,000 events) were collected and analyzed using an EPICS XL-MCL flow cytometer (Coulter, Hialeah, FL). Dead cells were excluded on the basis of forward and side scatter. Dot plot images were produced with the aid of WinMDI (version 2.1.2) by Joseph Trotter (Scripps Research Institute, La Jolla, CA). Abs used were FITC-labeled CD8 (53-6.7, PharMingen, San Diego, CA), PE-labeled CD8 (Caltag, South San Francisco, CA), FITClabeled CD8.2 (2.43 rat anti-mouse IgG2b affinity purified), FITC-labeled CD8.2 (53-5.8, PharMingen), CD8.1 ( , Serotec, Washington DC), PE-labeled goat anti-mouse IgG1 (Caltag), PE-labeled CD4 (Becton Dickinson), RED613-labeled CD4 (H129.19, Life Technologies), biotinylated anti-cd4 (GK1.5, affinity purified), Tricolor-labeled streptavidin (Caltag), and PE-labeled TCR- (H57-597, PharMingen). Results Expression of the CD8 (TL) construct in T cell hybridomas Deletion of the cytoplasmic domain of CD8 has been reported to result in impaired development of CD8 T cells. To examine the effect of this mutation on CD8 -associated Lck kinase activation,

4 The Journal of Immunology 185 FIGURE 1. Expression of CD8 (tailless) cdna. T cell hybridoma cells previously expressing human CD4 and human CD8 were transfected with either wild-type CD8 cdna (CD8 (wt)) or cytoplasmic domain-deleted CD8 (CD8 (TL)). Cells were stained with anti-tcr (F23.1), anti-cd4 (Leu 3a), anti-cd8 (Leu 2a), or anti-cd8 (2ST8-5H7). FITC-labeled goat anti-mouse Ig was used as a secondary Ab, and the cells were analyzed by flow cytometry. we generated murine T cell hybridomas that stably coexpress human CD4, CD8, and either wild-type CD8 (CD8 (wt)) or CD8 (TL), in which 17 of 19 amino acids in the cytoplasmic domain have been deleted. T cell hybridomas expressing CD8 (wt) or CD8 (TL) were derived by transfection of the parental murine BYDP hybridoma described previously (20, 21). For the T cell hybridomas, surface expression of the transfected human coreceptors was examined by flow cytometry using Abs to TCR (F23.1), CD4 (Leu 3a), CD8 (Leu 2a), or CD8 (2ST8-5H7; Fig. 1). While Leu 2a recognizes both the and forms of CD8, 2ST8-5H7 specifically recognizes the CD8 heterodimer. As dimerization is mediated by disulfide bonds involving cysteine residues in the extracellular region of CD8 and -, the cytoplasmic deletion was not expected to alter cell surface expression of the mutant CD8 (TL) heterodimer. Ab staining and analysis by flow cytometry confirmed that the heterodimers with wild-type and mutant CD8 were recognized equally well by Leu 2a and 2ST8-5H7 Abs. Clones expressing comparable levels of wild-type CD8 and mutant CD8 (TL) heterodimer were chosen for further analysis. Hybridomas expressing human CD4 and CD8 coreceptors served as additional controls. the presence of CD8 (TL) cannot be attributed to differences in the level of expression between the two cell lines, as CD8 and CD8 expression were comparable. Furthermore, the Lck kinase activity associated with CD4 was comparable in cells expressing the wild-type or mutant CD8 chain. Thus, deletion of the cytoplasmic domain of CD8 significantly reduces the enhancement in CD8 -associated Lck kinase activity observed with the wild-type molecule. Lck association with CD8 chain is decreased in hybridomas expressing CD8 or CD8 (TL) compared with CD8 (wt) Previously, we showed that the enhanced CD8-associated Lck kinase activity observed with coexpression of a wild-type CD8 chain was accompanied by a more stable association of Lck with the CD8 chain. Specifically, it was observed that when cells were lysed in 1% Nonidet P-40, a harsher detergent than Brij-96, upon CD8 immunoprecipitation, less Lck remained associated with CD8 than with CD8. The lack of CD8 -associated Lck kinase activation observed in cells expressing the cytoplasmic domain-deleted form of the CD8 chain could, therefore, be due in CD8 -associated Lck kinase activity is decreased in hybridomas expressing CD8 or CD8 (TL) compared with CD8 (wt) To assess the influence of the cytoplasmic domain of CD8 on CD8 -associated Lck kinase activity, in vitro kinase assays were performed with hybridoma cells expressing CD8 alone, CD8 (wt), or CD8 (TL). T cell hybridomas were stimulated at 37 C by Ab-mediated cross-linking of CD4 or CD8. After lysis in 1% Brij-96, in vitro kinase assays were performed. Ab-mediated cross-linking of CD8 on cells expressing the mutant CD8 (TL) heterodimer resulted in four- to sixfold weaker activation of Lck kinase activity compared with hybridomas expressing the wild-type CD8 heterodimer (Fig. 2). A similar reduction in Lck kinase activity was also observed in hybridomas expressing only the CD8 homodimer. The lack of enhancement of Lck kinase activity associated with CD8 in FIGURE 2. Comparison of CD8 -associated Lck activity in T cell hybridomas expressing wild-type or tailless CD8. Cells were stimulated for 3 min at 37 C with anti-cd4 or anti-cd8 Ab cross-linking. Cells were lysed in 1% Brij-96 detergent and immunoprecipitated with protein A- Sepharose beads, and in vitro kinase assays were performed for 3 min at 30 C. Proteins were resolved using 8% SDS-PAGE, transferred onto PVDF membranes, and developed by autoradiography. A representative experiment is shown. Phosphorimager analysis indicated a fourfold reduction in radioactive incorporation in the 56-kDa Lck band upon CD8 crosslinking of hybridomas expressing CD8 (TL) compared with CD8 (wt).

5 186 CD8 CYTOPLASMIC TAIL REGULATES Lck AND CD8 T CELL DEVELOPMENT FIGURE 3. Comparison of CD8-Lck association in cells expressing wild-type or tailless CD8. Cells were lysed with 1% Nonidet P-40. All lysates were immunoprecipitated with anti-cd4 or anti-cd8 Abs. The proteins were resolved by 10% SDS-PAGE and immunoblotted with anti- Lck Ab (C-terminus). The Ig chain is seen in some lanes below the Lck band. The same membrane was stripped and reblotted with anti-cd8 Ab. A representative experiment is shown. part to decreased association of Lck with the CD8 chain. Hybridomas expressing CD8, CD8 (WT), or CD8 (TL) were lysed in 1% Nonidet P-40. Lysates were immunoprecipitated with CD4 or CD8 Ab and were immunoblotted with anti-lck Ab. An approximately twofold reduction in the basal level of Lck kinase associated with CD8 was observed in cells expressing tailless CD8 compared with the wild-type molecule (Fig. 3). Western analysis of the same membrane with anti-cd8 Ab revealed comparable, if not greater, amounts of CD8 immunoprecipitated from lysates of hybridomas expressing CD8 (TL) and CD8 compared with CD8 (wt). Comparable amounts of Lck were found in association with CD4 in all cell lines. The decreased activation of Lck kinase activity following crosslinking of the mutant CD8 (TL) heterodimer may in part be due to the diminished association of Lck with CD8 chain seen in the presence of the mutant CD8 chain. However, since the difference in the amount of Lck protein associated with CD8 is twofold, whereas the difference in Lck kinase activity is four- to sixfold, CD8 may play an additional role in the efficient activation of CD8 -associated Lck kinase activity. It has been observed that upon activation, Lck may form dimeric or multimeric complexes, which may be necessary for optimal activation of kinase activity (23). It is possible that a mutant CD8 (TL) heterodimer may interfere with the formation of these complexes as well as directly alter the association of Lck with the CD8 chain. Tyrosine phosphorylation of intracellular substrates is also diminished in cells expressing the mutant CD8 (TL) molecule The lack of CD8 -associated Lck kinase activation observed with expression of the mutant CD8 molecule also correlated with lack of enhanced tyrosine phosphorylation of several intracellular substrates. Hybridomas expressing CD8 (wt) or CD8 (TL) were stimulated by cross-linking the TCR alone or along with CD8 or CD4. All cell lines express CD4, and stimulation via CD4 served as a positive internal control for stimulation. After stimulation and lysis, the lysates were immunoprecipitated with an anti-phosphotyrosine Ab 4G10 and subsequently immunoblotted with a second anti-phosphotyrosine Ab, RC-20H. The pattern of tyrosine-phosphorylated proteins was compared between the cell lines. Despite comparable enhancement of tyrosine phosphorylation of several proteins upon TCR/CD4 cross-linking in all cell lines, a distinct difference in the phosphorylation patterns was observed upon TCR/CD8 cross-linking. In cells expressing the mutant CD8 (TL) chain, minimal enhancement of tyrosine phosphorylation was observed compared to that in cells expressing the wild-type molecule (Fig. 4). This lack of enhancement correlates FIGURE 4. Comparison of tyrosine phosphorylation of intracellular proteins after TCR/CD8 cross-linking of cells expressing wild-type or tailless CD8. Cells were stimulated at 37 C for 3 min with anti-tcr Ab cross-linking alone or in combination with anti-cd4 or anti-cd8 Ab. Cells were lysed and immunoprecipitated with anti-phosphotyrosine Ab (4G10) and protein A-Sepharose beads preincubated with RAMG. The bound proteins were eluted using 10 mm p-nitrophenylphosphate, resolved by 10% SDS-PAGE, immunoblotted with anti-phosphotyrosine Ab (RC20H), and developed by ECL. Several proteins were differentially phosphorylated with expression of wild-type CD8, but not with the cytoplasmic-domain deleted mutant CD8, as indicated by the arrows. with the lack of activation of CD8 -associated Lck kinase activity observed in in vitro kinase assays of lysates derived from cells expressing the cytoplasmic domain-deleted form of CD8 and suggests that several downstream, activation-dependent events may be altered by expression of this mutation. CD8-associated Lck kinase activity is decreased in CD8 / and CD8 (TL) mice We have previously shown that expression of a tailless form of CD8 in thymocytes of transgenic mice inhibits CD8 T cell development by dominantly inhibiting endogenous CD8 function. To determine whether this developmental block correlates with a defect in Lck kinase activation, we examined CD8-associated Lck activity from CD8 (TL) transgenic mice. For comparison, we also examined mice with a germline disruption of the CD8 gene (CD8 / ) and mice that express the CD8 (TL) transgene and are heterozygous for endogenous CD8 (CD8 / ). We have been unable to obtain CD8 (TL) transgenic mice on a CD8 / background due to the tight linkage of the transgene to the endogenous CD8 locus (unpublished observations). The effect of CD8 mutations on CD8-associated Lck kinase activity in thymocytes parallels the effect seen with T cell hybridomas. Thymocytes from CD8 / mice display a sixfold reduction of CD8-associated Lck activity compared with wild-type mice, whereas thymocytes from CD8 / CD8 (TL) transgenic mice and CD8 / CD8 (TL) mice have four- and twofold reductions in CD8-associated Lck activity, respectively (Fig. 5). In two experiments, we included enolase as an exogenous substrate for Lck. The normalized Lck activity from quantification of the enolase bands corresponded with values obtained from quantification of the Lck bands. Although thymocytes from CD8 / mice have reduced surface expression of CD8 (13 15) (Fig. 6, A and B), changes in CD8 levels cannot fully account for the decrease in CD8-associated Lck activity, since thymocytes from CD8 (TL) transgenic mice also display reduced Lck activity and have surface

6 The Journal of Immunology 187 FIGURE 5. CD8-associated Lck kinase activity in CD8 mutant mice. Thymocytes from the indicated mice were stimulated by Ab-mediated cross-linking of either CD4 or CD8. In vitro kinase assays were performed on the immunoprecipitates, and proteins were resolved by 8.5% SDS- PAGE. Radioactivity was quantitated using a phosphorimager, and in each experiment, Lck activity was normalized to the value for wild-type mice. A representative experiment is shown. The results of four experiments were averaged. Normalized Lck activities associated with CD4 were for CD8 /, for CD8 / CD8 (TL), and for CD8 / CD8 (TL). Lck activities associated with CD8 were in CD8 /, in CD8 / CD8 (TL), and in CD8 / CD8 (TL). FIGURE 7. Amount of Lck protein associated with CD8 in wild-type and mutant CD8 mice. Thymocytes were lysed with either 1% Brij 97 or 1% Nonidet P-40 and immunoprecipitated with anti-cd4 or anti-cd8 covalently bound to Sepharose-protein G beads. Immunoprecipitates were resolved by 10% SDS-PAGE and were analyzed by Western blot analysis using anti-lck Abs. Lck bands were quantitated by densitometry, and values for each sample were normalized to CD8 immunoprecipitates for wildtype thymocytes run in the same experiment. The normalized CD8-associated Lck values averaged from six experiments are for CD8 /, for CD8 / CD8 (TL), and for CD8 / CD8 (TL). A representative experiment is shown. levels of CD8 comparable to those in wild-type mice (Fig. 6, A and B). Comparable Lck activity is associated with CD4 in all the strains examined, indicating that CD8 mutations do not cause a global reduction of Lck activity. Thus in thymocytes, as in T cell hybridomas, the cytoplasmic domain of CD8 increases the Lck activity associated with CD8. While thymocytes from both CD8 / and CD8 (TL) transgenic mice have reduced CD8 -associated Lck activity, this effect is more dramatic in CD8 / mice. This might result from a partial contribution to CD8 -associated Lck activity by the tailless form of CD8. Alternatively, this difference could be attributed to the presence of endogenous, wild-type CD8 in the CD8 (TL) transgenic mice. To examine this question, we compared the surface expression of endogenous CD8 in thymocytes from CD8 /, CD8 / CD8 (TL) transgenic, and CD8 / CD8 (TL) transgenic mice (Fig. 6C). We find a good correlation between the surface expression of endogenous, wild-type CD8 and the CD8- associated Lck activity between thymocytes from these different genotypes. This correlation suggests that the difference in CD8- associated Lck activity between the CD8 / mice and the CD8 (TL) transgenic mice is mainly the activity contributed by the presence of endogenous CD8 in the latter. This, in turn, implies that Lck activation by CD8 can be attributed primarily to its intracellular domain. Lck association in CD8 / and CD8 (TL) mice Analysis of T cell hybridomas indicates that CD8 can stabilize the association between CD8 and Lck. To determine whether the same is true in thymocytes, we examined the amount of Lck protein associated with CD8 in thymocytes of the various mice by Western blot analysis using anti-lck Abs. Lysates of unstimulated thymocytes were immunoprecipitated with anti-cd4 or anti-cd8 Abs. Despite the dramatic decrease in CD8 -associated Lck kinase activity in CD8 / or CD8 (TL) mice compared with that in wild-type animals, significant amounts of Lck were still detected in CD8 immunoprecipitates of thymocytes of the various mutant mice, although in some experiments a slight decrease in CD8 associated Lck levels was observed (Fig. 7). The results suggest that the effect of CD8 on CD8 -associated Lck kinase activity in thymocytes cannot be due solely to a change in the amount of associated Lck, but must also be due to a change in the specific activity of Lck. FIGURE 6. Expression level of surface CD8 in CD8 (TL) transgenic mice. A, Thymocytes from wild-type, CD8 /, CD8 (TL) transgenic mice with one or two copies of endogenous CD8 were stained with fluorescent Abs and analyzed by flow cytometry. Mode fluorescence values were then normalized to wild-type mice that were analyzed in the same experiment. The bars represent the mean, and the dots are values from individual mice. Representative histograms of CD8 (B), endogenous CD8 (CD8.2; C), and transgenic CD8 (TL) (CD8.1; D) from the indicated mice are shown: spectrum a, wild-type; spectrum b, CD8 / ; spectrum c, CD8 / CD8 (TL); and spectrum d, CD8 / CD8 (TL).

7 188 CD8 CYTOPLASMIC TAIL REGULATES Lck AND CD8 T CELL DEVELOPMENT FIGURE 8. Thymic subsets in mice expressing CD8 (TL). A, The percentage of mature CD4 and CD8 thymocytes from wild-type, CD8 /, and CD8 (TL) transgenic mice with either one or two copies of endogenous CD8. The bars represent the mean percentage from each set of mice; values from individual mice are superimposed as dots. Mean percentages for CD4 thymocytes were % in wild-type mice, % in CD8 /, % in CD8 / CD8 (TL), and % in CD8 / CD8 (TL). For CD8 thymocytes, these values were % in wild-type, % in CD8 /, % in CD8 / CD8 (TL), and % in CD8 / CD8 (TL). B, Representative data from three-parameter flow cytometric analysis. Thymocytes were stained for surface expression of CD4, CD8, and TCR- using fluorescent Abs. The numbers inside the quadrants represent the percentage of cells in each population. Mature thymocytes have high levels of TCR and are either CD4 CD8 or CD4 CD8. Decreased CD8 -associated Lck kinase activity correlates with impaired CD8 T cell development The effect of the CD8 mutations on the development of CD8- lineage T cells correlates with the reduction of CD8-associated Lck activity. Thymocytes and lymph node cells from CD8 /, CD8 / CD8 (TL) transgenic, and CD8 / CD8 (TL) transgenic mice were analyzed by flow cytometry for expression of TCR, CD4, and CD8. CD8 / mice exhibited a fivefold reduction in the percentage of mature CD8 lineage thymocytes, whereas CD8 / CD8 (TL) transgenic and CD8 / CD8 (TL) transgenic mice had three- and twofold fewer CD8 lineage thymocytes compared with wild-type animals. In contrast, the percentage of mature CD4 lymphocytes was not affected by any of these mutations (Fig. 8). A similar trend was seen among lymph node T cells from the CD8 mutants. The CD8 / mice had the most severe phenotype, exhibiting a CD8 to CD4 ratio 6.6-fold less than that of the wild-type animals. The CD8 / CD8 (TL)mice and CD8 / CD8 (TL) mice had respective ratios that were 2.2- and 1.8-fold less than that of the wild-type

8 The Journal of Immunology 189 FIGURE 9. Flow cytometric analysis of peripheral T cells in CD8 (TL) transgenic mice. A, The ratio of mature CD8 to CD4 T cells in lymph nodes. The genotype of the mice is indicated. Bars are the average of the ratios for each set of mice, and superimposed dots are values from individual mice. Percentage means of CD8 to CD4 ratio are as follows: wild-type, %; CD8 /, %; CD8 / CD8 (TL), %; and CD8 / CD8 (TL), %. B, Flow cytometric analysis of total lymph node cells stained for expression of CD4 and CD8. The numbers inside the quadrants represent the percentage of cells in each population. TCR- CD8 lymphocytes are TCR and NK1.1 negative (data not shown). Parallel analysis indicates that the percentage of CD4 cells in splenocytes is not affected by the CD8 mutation, suggesting that the effect on the CD8 to CD4 ratio is a result of a decrease in the number of CD8 cells. animals (Fig. 9). Examination of these results revealed a correlation between the developmental defects in these mice and the corresponding Lck kinase activity. This correlation suggests that the decrease in Lck activity in the CD8 mutant mice may account for the loss of mature CD8-lineage T cells in these mice. Discussion There is growing evidence supporting a unique role for CD8 in both MHC class I Ag recognition and T cell activation that may enable CD8 heterodimers to function more efficiently than CD8 as a coreceptor for the TCR complex. Although recent reports suggested that the ability of CD8 to function more efficiently than CD8 depends on enhanced recognition and binding to Ag, our present study supports the view that the signaling capabilities of CD8 are also critical. We have shown that one of the biochemical events that is altered by lack of expression of CD8 or expression of the cytoplasmic domain-deleted CD8 chain is the activation of Lck kinase. CD8 enhances the activity of CD8 -associated Lck kinase, and the present study revealed that the cytoplasmic domain of CD8 is critical for this enhancement. The Lck kinase activity stimulated by the cytoplasmic domain of CD8 resulted in enhanced cellular tyrosine phosphorylation and may thus be critical in coupling the CD8 coreceptor to downstream signaling pathways. The enhanced CD8 -associated Lck activity mediated by the cytoplasmic domain of CD8 was shown to result from both 1) a more stable association of Lck with CD8 and 2) an enhanced specific enzymatic activity. The cytoplasmic domain of CD8 may make the Lck binding site of CD8 more accessible. Alternatively, although there are no known protein binding motifs in the short cytoplasmic domain, associated proteins may play a role in stabilizing the CD8 -Lck association and/or enhancing Lck kinase activity. The greater reduction observed in kinase activity compared with the level of Lck association with various CD8 mutations suggested that CD8 also influences the kinase activity of CD8 associated Lck. Activation of Lck kinase activity requires several steps, including phosphorylation, dephosphorylation, and conformational changes that lead to formation of the active catalytic site (reviewed in Ref. 24). Dimerization, and possibly multimerization, of Lck may also be required for full activity. The cytoplasmic domain of wild-type CD8 chain, either directly or indirectly, may regulate activity by influencing any of these steps. The phenotypes of the various CD8 mutant mice suggested that CD8 plays a critical role in the selection and differentiation of CD8 T cells. CD8 -deficient mice and transgenic mice expressing a cytoplasmic tail-deleted CD8 exhibited five- and twofold fewer mature CD8 T cells, respectively (13, 18). When CD8 / mice were crossed with mice expressing the H-Y-specific TCR transgene, a 20-fold reduction in the number of mature CD4 CD8 thymocytes was observed (13). As the number of double-positive cells was normal, this observation pointed to a specific role of CD8 in positive selection. The developmental

9 190 CD8 CYTOPLASMIC TAIL REGULATES Lck AND CD8 T CELL DEVELOPMENT deficiencies observed in the CD8 tailless transgenic mice suggested that a critical component of the function of CD8 in CD8 T cell development is the generation of signals that direct positive selection, since CD8 heterodimers consisting of either wild-type or tailless CD8 would be expected to make indistinguishable extracellular contacts with the selecting ligand(s). Indeed, CD8 -associated Lck kinase activity was dramatically decreased in both CD8 -deficient mice and CD8 tailless transgenic mice. By generating CD8 tailless transgenic mice with reduced levels of endogenous CD8 expression, we have also been able to establish a correlation between decreased CD8 -associated Lck kinase activity observed with CD8 mutations and impairment of CD8 T cell development. CD8 -deficient mice that do not express any endogenous CD8 exhibited the most severe impairment in CD8 T cell development and also exhibited, on the average, the most dramatic reduction in CD8 -associated Lck activity, relative to wild-type mice. Comparison of the CD8 tailless transgenic mice that are either homozygous or heterozygous for endogenous CD8 expression revealed that reduction in endogenous CD8 expression resulted in both 1) additional impairment of CD8 T cell development and 2) a further decrease in CD8 -associated Lck activity. Therefore, while we do not exclude the possibility that other changes in activation and signaling may contribute to the developmental impairment observed in CD8 / or CD8 (TL) mice, our data support the importance of the Lck regulatory capability of the cytoplasmic domain of CD8 in differentiation of CD8-lineage cells. Appropriately regulated Lck kinase activity is necessary at multiple stages of T cell development, as suggested by studies of mice lacking Lck expression and transgenic mice expressing catalytically inactive or constitutively active Lck constructs (25 28). Since the defect in T cell development observed in mice bearing a homozygous disruption of CD8 or in mice expressing the CD8 (TL) transgenic construct occurs subsequent to the doublepositive stage, the enhanced Lck activity contributed by the CD8 chain may be especially important for efficient positive selection of CD8 T cells. A recent study established a role for Lck kinase activity in the positive selection of double-positive thymocytes; mice expressing a catalytically inactive Lck transgene under the control of the distal Lck promoter exhibited a twofold reduction in positive selection of the H-Y-specific TCR (29). The results of studies with transgenic mice expressing signaling molecules thought to be downstream of Lck kinase activation were also consistent with a critical role for Lck in positive selection; transgenic mice expressing H-ras N17, a dominant negative mutant of Ras, or a catalytically inactive form of MEK-1 (a mitogen-activated protein kinase kinase) were impaired in their ability to positively select CD8 thymocytes expressing the H-Y-specific TCR transgene (30, 31). The importance of CD8-associated Lck kinase activity for optimal positive selection and development of CD8 T cells has been suggested by several studies. Overexpression of a CD4 transgene inhibited the positive selection of a class I MHC-restricted TCR (32). In this model, the association of Lck with the cytoplasmic domain of CD8 was decreased, presumably from competition with the cytoplasmic domain of CD4. Introduction of a cytoplasmic domain-deleted CD8 chain as a transgene into CD8-deficient mice also resulted in decreased numbers of mature CD8 T cells compared with those in wild-type control mice (33). In contrast, positive selection appeared to proceed at significant levels in transgenic mice expressing both a class I MHC-restricted TCR and a CD8 molecule with point mutations in the Lck binding site (34). However, compared with wild-type mice, the efficiency of this selection was decreased. Thus, CD8-associated Lck activity may be critical for optimal efficiency of CD8 T cell positive selection. Our studies are consistent with the growing evidence suggesting the importance of the signaling capabilities of the CD8 coreceptor in T cell maturation and differentiation. Both CD8 and CD4 serve dual roles as adhesion molecules and signaling receptors. CD8 increases the avidity of the interaction between the TCR and class I MHC molecule found on the T cell and the APC, respectively. However, recent measurements of the CD8/MHC interaction indicate that the binding of CD8 and CD8 is similar (35). Therefore, it is unlikely that the reduction in CD8 T cells observed in CD8 / and CD8 (TL) mice is primarily due to decreased interaction with the MHC/peptide complex. The cytoplasmic domains of the CD8 heterodimer must also transduce signals, including activation of Lck kinase, that are critical for normal CD8 T cell development. Future studies will aim to elucidate the identity and specificity of these signals. Acknowledgments We thank Dr. Ellis Reinherz (Dana-Farber Cancer Institute) for providing the 2ST8-5H7 Ab, Dr. James Allison (University of California, Berkeley) for providing the GK1.5 and hybridomas, and Dr. David Raulet (University of California, Berkeley) for providing the RL172.4 and hybridomas. References 1. Dembic, Z., W. Haas, R. Zamoyska, J. R. Parnes, M. Steinmetz, and H. von Boehmer Transfection of the CD8 gene enhances T cell recognition. 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