Proc. Natl. Acad. Sci. USA Vol. 91, pp. 6933-6937, July 1994 Immunology Engagement of the external domains of CD45 tyrosine phosphatase can regulate the differentiation of immature CD4+CD8+ thymocytes into mature T cells (trosine kinase/posdve selectlon/thymus) PATRICIA BENVENISTE, YousuKE TAKAHAMA*, DAVID L. WIEST, TOSHINORI NAKAYAMAt, SUSAN 0. SHARROW, AND ALFRED SINGER* Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by David W. Talmage, March 25, 1994 ABSTRACT Immature precursor cells are induced in the CD4+CD8+ thymocytes to undergo positive selection and to thymus to express clonotypic T-cell antigen receptors (TCRs) differentiate into mature SP T cells. and to differentiate into mature T cells. Perhaps the least understood event which occurs during intrathymic develop- ment is the positive selection of immature CD4+CD8+ thymo- MATERIALS AND METHODS cytes for differentiation into mature CD4+ and CD8+ T cells Monoclonal Antibodies (mAbs) Used for Injection. Rat IgG based on the TCR specificity individual thymocytes express. mAbs for injection were purified from supernatants of the TCR expression by CD4+CD8+ thymocytes is quantitatively following cell lines: M1/9.3, anti-CD45 rat IgG2a (14); 2.43, regulated by CD4-mediated activation of p56k' protein- anti-CD8 rat IgG2b (15); GK1.5, anti-CD4 rat IgG2b (16). tyrosine kinase whose activity can in turn be regulated by the Animals and In Vivo Injection. CS7BL/6 newborn mice membrane-bound protein-tyrosine-phosphatase CD45. Here were injected daily with mAb in amounts that had been found we show that antibody engagement of CD45 external dain to be saturating of both peripheral and intrathymic sites, enhances Lck tyrosine kinase activity in CD4+CD8+ thymo- which for mAbs to CD45 and CD8 involved injection with 500 cytes, inhibits TCR expression, and inhibits differentiation of jug ofpurified mAb on days 1-5, 1 mg ofpurified mAb on days immature CD4+CD8+ thymocytes into mature T cells. Thus, 6-11, and 1.5-2.5 mg of purified MAb on days 12-21. Cells engagement of the external domains of CD45 tyrosine phos- were assessed 1 day after the last injection. phatase can regulate the ability of immature CD4+CD8+ Cell Isolations. CD4+CD8+ thymocytes from mice injected thymocytes to undergo positive selection, suggesting an impor- with either saline or anti-CD45 mAb were isolated by panning tant regulatory role for intrathymic ligands that are capable of on plates coated with IgM mAb to CD8.2. Because engaging CD45 within the thymus. CD4+CD8+ thymocytes from mice treated in vivo with rat IgG anti-CD8 mAb 2.43 were coated with antibody and had Immature CD4+CD8+ thymocytes express low levels of no free CD8 sites available, CD4+CD8+ thymocytes from T-cell antigen receptors (TCRs) that are increased during these mice were isolated by panning on plates coated with positive selection (1-3). TCR expression in immature goat anti-rat IgG. The isolated populations were >96% CD4+CD8+ thymocytes is low in part because it is quanti- CD4+CD8+ cells. tatively inhibited by activation ofthe protein-tyrosine kinase Immune-Complex Kinas Assay. The assay was performed p561ck (Lck), which is preferentially associated in CD4+CD8+ as described (4). Briefly, freshly isolated CD4+CD8+ thymo- thymocytes with the cytoplasmic tail of surface CD4 mole- cytes were lysed at 108 per ml in 1% (wt/vol) Triton X-100 cules (4). Surface CD4 molecules on CD4+CD8+ thymocytes lysis buffer (50 mM Tris, pH 7.4/0.15 M NaCl/1 mM are chronically engaged by major histocompatibility complex Na3VO4/2 mM EDTA with aprotinin at 20 jug/ml and leu- class II molecules in the thymus, resulting in the continuous peptin at 10 tg/ml) for 20 min at 40C. Clarified post nuclear activation of CD4-associated Lck molecules in CD4+CD8+ supernatants were agitated at 40C for 1-2 hr with anti-CD4 thymocytes and, consequently, low TCR expression (4-8). It mAb preadsorbed to protein G-Sepharose, following which is not known how Lck tyrosine kinase activity is down- the extracts were reprecipitated with rabbit anti-Lck (anti- regulated in developing CD4+CD8+ thymocytes to permit the body 688, generously provided by L. E. Samelson, National increased TCR expression associated with positive selection. Institutes of Health, Bethesda, MD) bound to protein However, it is known that Lck tyrosine kinase activity can be A-Sepharose. Beads were washed three times in lysis buffer regulated in mature T cells by the transmembrane protein- lacking EDTA and incubated for 3 min at room temperature tyrosine-phosphatase CD45 (9-13), which dephosphorylates in kinase buffer [20 mM Hepes, pH 7.5/100 mM NaCl/5 mM the regulatory Tyr-505 residue in the Lck kinase domain. MgCl2/5 mM MnCl2/2 AuM Na2ATP with 15 uCi of Consequently, it seemed possible that CD45, which is ex- [y-32P]ATP per reaction (7000 Ci/mmol; 1 Ci = 37 GBq)]. pressed at high levels on all thymocytes, would regulate Lck Kinase reactions were quenched with sample buffer and activity in developing CD4+CD8+ thymocytes and, as a resolved in SDS/8% polyacrylamide gels. To remove free result, regulate the ability of immature CD4+CD8+ thymo- 32p, gels were equilibrated against five to six changes of 10%o cytes to differentiate into mature CD4+ and CD8+ "single- positive" (SP) T cells. Indeed, the present study demon- Abbreviations: mAb, monoclonal antibody; SP, single-positive; strates that engagement of the external domains of CD45 TCR, T-cell antigen receptor. tyrosine phosphatase can regulate the ability of immature *Present address: Institute of Immunology, Syntex Research, Nii- hari, Ibaraki 300-41, Japan. tPresent address: Department ofImmunology, Faculty ofMedicine, The publication costs ofthis article were defrayed in part by page charge University of Tokyo, Tokyo, Japan. payment. This article must therefore be hereby marked "advertisement" *To whom reprint requests should be addressed at: National Cancer in accordance with 18 U.S.C. §1734 solely to indicate this fact. Institute, Building 10, Room 4B-17, Bethesda, MD 20892. 6933 Downloaded by guest on October 2, 2021 6934 Immunology: Benveniste et al. Proc. Nad. Acad Sci. USA 91 (1994) methanol/1Oo acetic acid for a total of 6 hr, dried, and Ab: anti-CD4 - anti-Lck autoradiographed at -800C. Immppt. Anti-Lck Imnunoblottng. Samples for anti-Lck immuno- U) L) blotting were boiled for 3 min in SDS sample buffer, resolved C C: by SDS/PAGE, and blotted onto nitrocellulose. Following In Vivo _ L ; transfer, blots were blocked for 1 hr at room temperature with MAb Treatment: co o M C clc 5% milk protein in phosphate-buffered saline and then probed for 2 hr with 1:200 anti-Lck antibody 688 diluted in phos- Immune Complex phate-buffered saline containing 5% milk protein. Bound antibody was visualized with 125I-labeled protein A by auto- Lck - Kinase Assay radiography. Flw Cytometry. Flow cytometry was performed with Enolase - either a modified dual-laser FACS II or a modified dual-laser FACStar PLUS (Becton Dickinson). One-color anti-CD3e analyses were performed using 145-2C01 mAb directly con- jugated with fluorescein isothiocyanate (FITC) (PharMin- gen). For two-color analyses, cells either were reacted with FITC-anti-CD4 (Rm4-5, PharMingen) followed by biotin- Lck-lUi Immunoblot labeled anti-CD8 (53-6-72, Becton Dickinson Immunocytom- etry Systems) and Texas Red-streptavidin (BRL) or were reacted with FITC-anti-TCR.8 (H57-597) followed by biotin- Blotting Ab: anti-Lck labeled anti-CD5 and Texas Red-streptavidin. For three- coloranalyses, cells were reacted with FITC-T3.70 (prepared FIG. 1. Effect of in vivo antibody treatments on CD4-asiated in our followed Lck activity and abundance in CD4+CD8+ thymocytes. Freshly laboratory), by R-phycoerythrin-conjugated isolated CD4+CD8+ thymocytes from C57BL/6 mice that had been anti-CD4 (GK1.5, BDIS), biotin-anti-CD8, and Texas Red- injected daily from birth with saline, IgG mAb to CD45 (M1/9.3), or streptavidin. IgG mAb to CD8 (2.43) as indicated were compared at 3 weeks ofage All fluorescence data were collected with logarithmic am- for Lck activity (Upper) and abundance (Lower). Activity of CD4- plification on 50,000-250,000 viable cells as determined by associated Lck (lanes 1-3) was determined by assessing anti-CD4 forward light scatter intensity and propidium iodide exclu- immunoprecipitates ofcell lysates (20 x 106 cell equivalents perlane) sion. in an immune-complex kinase assay (Upper), whereas abundanc of CD4-associated Lck protein was quantitated by immunoblotting anti-CD4 immunoprecipitates (6 x 106 cell equivalents per lane) with RESULTS anti-Lck antibody 688 (Lower). Residual Lck that was not immuno- precipitated by anti-CD4 mAb was isolated by reimmunoprecipitat- We began the present study by examining the effect of ing remaining cell lysates with anti-Lck antibody (lanes 4 and 5). antibody engagement of the external domains of CD45 on CD4-associated Lck tyrosine kinase activity in normal crosslinking mAb to CD4 to disrupt intrathymic CD4-ligand CD4+CD8+ thymocytes. Although thymocytes express mul- interactions), both ofwhich increase TCR expression among tiple CD45 isoforms (17-20), the present study utilized an IgG CD4+CD8+ thymocytes. Interestingly, antibody engagement mAb to CD45 (M1/9.3) that is reactive with all CD45 isoforms of CD45 inhibited TCR increases resulting from CD4 disen- (14). Newborn mice were injected daily with saline, IgG mAb gagement (Fig. 2). First, as assessed in vitro, mAb to CD45 to CD45, or IgG mAb to CD8 (2.43) (15) until intrathymic inhibited the upregulation ofTCR by CD4+CD8+ thymocytes binding was detected, which for anti-CD45 required that during 3TC single-cell suspension cultures (Fig.