Proc. Natl. Acad. Sci. USA Vol. 91, pp. 6933-6937, July 1994 Engagement of the external domains of CD45 tyrosine phosphatase can regulate the differentiation of immature CD4+CD8+ into mature T cells (trosine kinase/posdve selectlon/) 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. 2A Left). peripheral binding sites first be saturated. Consequently, mAb to CD45 was found to interfere with TCR CD4+CD8+ thymocytes were obtained from injected animals upregulation at 3 weeks ofage, at whichtime intrathymic anti-CD45 binding whether the mAb was added directly to suspension cultures had achieved saturation and CD45 expression was only min- or was previously injected in vivo, and was specific in that imally reduced. Lysates of CD4+CD8+ thymocytes from all mAb to CD8 had no effect (Fig. 2A Left). The inhibition of three experimental groups were found to have similar amounts TCR upregulation by CD45 engagement was a consequence of CD4-associated Lck protein (Fig. 1 Lower) but different of its regulation of tyrosine kinase activity, as its effect was amounts of CD4-associated Lck activity (Fig. 1 Upper). The abrogated by herbimycin A, which targets tyrosine kinases activity of CD4-associated Lck in immune-complex kinase such as Lck for degradation (Fig. 2A Right) (22). Second, as assays was significantly higher in CD4+CD8+ thymocytes assessed in vivo, injection ofmAb to CD45 did not itselfalter from anti-CD45-treated mice, as indicated both by Lck auto- TCR levels (Fig. 2B, lanes 1 and 3), but it did significantly phosphorylation and bytransphosphorylation ofan exogenous interfere with TCR increases resulting from CD4 disengage- substrate, enolase (Fig. 1 Upper). Residual Lck activity not ment induced by injection of mAb to CD4 (Fig. 2B, lanes 2 associated with CD4 was unaffected by anti-CD45 treatment and 4). In this experiment, increases in surface TCR levels (Fig. 1 Upper Right). Thus, in vivo antibody engagement of were similarly affected, but total cellular TCR levels are CD45 specifically enhanced CD4-associated Lck activity in displayed to demonstrate that CD45 engagement interfered developing CD4+CD8+ thymocytes. with TCR expression and did not simply induce internaliza- Because CD4-associated Lck activity regulates TCR ex- tion of surface TCR complexes. Thus, antibody engagement pression in CD4+CD8+ thymocytes (4-8, 21), we wished to of CD45 on CD4+CD8+ thymocytes enhanced CD4- examine whether antibody engagement of CD45 on associated Lck activity and interfered with upregulation of CD4+CD8+ thymocytes affected either TCR expression or its TCR expression resulting from CD4 disengagement. regulation by CD4. We have previously shown (4) that CD4 Since antibody engagement of CD45 interfered with ex- disengagement interrupts the continuous activation of CD4- perimentally induced upregulation of TCR expression in associated Lck in CD4+CD8+ thymocytes, permitting TCR CD4+CD8+ thymocytes, we thought that CD45 engagement expression to increase. CIX disengagement can be experi- might also affect the ability of immature CD4+CD8+ thymo- mentally induced in vitro (by placement in 3TC suspension cytes to differentiate into mature T cells, as this step is cultures to physically remove CD4+CD8+ thymocytes from associated with increased TCR expression (3). Neonatal mice intrathymic CD4 ligands) or in vivo (by injection of non- were injected daily with mAb to CD45 and assessed at 3 Downloaded by guest on October 2, 2021 Immunology: Benveniste et al. Proc. Natl. Acad. Sci. USA 91 (1994) 6935 A M ce,d

olAh added in) vitro i ",-, .S iIIi .1. ..., ., Afar B a nti-C1145 / Wr-.k. By " hi.-{ / 'I CZ~~~~~~~~~tD . CO~~~~~CCD 1 ''1 - '-1 , --- -- t Ct Ct 5:I6. UO anti CD8 kDa

- 200 11kr'. I.l! *. *a .- - 97 mlAb Injected TCRaJ3 - 69 ifI Vivo - 46 a nti-CD45

- 1 2 3 1 2 3 30 o s co o Log1o CD3 FLliorescence 'b.,cb D.t FiG. 2. Antibody engagement of CD45 inhibits TCR upregulation resulting from CD4 disengagement. Effect of anti-CD45 treatment on in vitro CD4 disengagement. (A) CD4+CD8+ thymocytes (107) from young adult mice were placed in single-cell suspension cultures for 14 hr in the absence orpresence ofherbimycin A (1 pM) and then assessed for surface TCR expression by staining with mAb to CD3e(145-2C11). (Upper) Cultures were supplemented with mAb to CD45 (M1/9.3) or IgM mAb to CD8 (3.155) as indicated. Each panel displays four curves: shaded curve, control staining with a negative control antibody (Leu-4 mouse anti-human antibody); long-dashed line, CD3 staining of cells cultured at 40C; short-dashed line, CD3 staining ofcells cultured at 37C; solid line, CD3 staining ofcells cultured at 37C with mAb. (Lower) CD4+CD8+ thymocytes were cultured overnight in medium or herbimycin but were obtained from mice that had been injected with either mAb to CD45 (1 mg ofpurified M1/9.3 antibody per mouse per day) or saline. Each panel displays four curves: shaded curve, control staining with a negative control antibody (Leu-4 mouse anti-human antibody); long-dashed line, CD3 staining of cells cultured at 4"C; short-dashed line, CD3 staining of cells from saline-injected mice cultured at 370C; solid line, CD3 staining of cells from mAb-injected mice cultured at 37C. TCR expression in 40C cultured CD4+CD8+ thymocytes from saline- and mAb-treated mice was identical. An equivalent amount of dimethyl sulfoxide, the solventforherbimycin A, was added to control "media" cultures. (B) Effect ofanti-CD45 treatment on in vivo CD4 disengagement. Total cellular TCR levels in freshly isolated CD4+CD8+ thymocytes were compared from C57BL/6 mice that had been injected with saline, IgG mAb to CD4 (GK1.5), IgG mAb to CD45 (M1/9.3), or a combination ofboth mAbs. Cell lysates were immunoprecipitated with anti-CD3e (145-2C01), resolved by SDS/13% PAGE undernonreducing conditions, and immunoblotted with mAb to TCR a chain (H28-710). Numbers below each lane represent relative band intensities as determined by densitometry. weeks ofage, at which time they normally contain significant pleted of mature T cells (data not shown). To more closely numbers of mature SP thymocytes and mature peripheral T examine the effect of anti-CD45 engagement on the genera- cells. In contrast to control mice, thymuses of treated mice tion of mature SP thymocytes, we performed experiments were essentially devoid of mature SP thymocytes, although utilizing fetal thymic organ cultures. Day 15 (Exp. 1) and day they did contain immature CD4-CD8- and CD4+CD8+ thy- 16 (Exp. 2) fetal thymic lobes were cultured for 7 days, mocytes (Fig. 3 Left). The absence ofmature SP thymocytes sufficient time to permit significant numbers of developing was not due to more efficient export to the periphery, as thymocytes to complete their developmental program and to peripheral lymphoid organs in these animals were also de- differentiate into phenotypically mature SP cells (Fig. 3 3 wk Thymus FTOC FTOC Exp. 1 Exp.2 Exp.2 mAb injected mAb 2 86 added 5 60 4 56 in Vivo (day 1-2 1) to FTOC -wX Saline Saline i 14 20 - as- e - co LO 00 5 7 0 2___ 2'20 0 CD1 2 87 6 62 6 51 70 2

anti-CD45 j anti-CD45

10' 1 5CD S.:27' ° E e \ , CD4 CD4 TCRj1 TCR1

FIG. 3. Treatment with mAb to CD45 inhibits the generation of SP thymocytes in normal and TCR transgenic mice. (Left) Mice were injected daily from birth with saline or IgG anti-CD45 mAb. Two-color fluorescence analysis for CD4 and CD8 expression was performed on thymocytes from mice in each group at 3 weeks of age. Numbers within each box ofcontour diagrams indicate the frequency ofcells within that box. (Center and Right) Fetal thymus organ cultures (F7OC) were performed on day 15 (Exp. 1) and day 16 (Exp. 2) fetal thymuses for 7 days. On days 0, 3, and 6 of the cultures, 0.1 ml of medium was aspirated, and 0.1 ml of either diluted saline or IgG anti-CD45 mAb (40 jg/ml) was added to the cultures. On day 7, thymic lobes (10-20 lobes per group) were gently teased and cells were analyzed with two-color flow cytometry using mAbs with indicated specificities. Right panels display single-color histograms of staining with FITC-labeled mAb to TCR 8 chain (H57-597; solid lines) or control mAb (shaded dash lines). Cell recoveries from FTOC in the presence of anti-CD45 mAb were 50% (Exp. 1) and 56% (Exp. 2) of those from control saline-treated FTOC. Downloaded by guest on October 2, 2021 6936 Immunology: Benveniste et al. Proc. Natl. Acad. Sci. USA 91 (1994)

Control Anti-CD45 (day 1 - day 21) that intrathymic positive selection ofCD4+CD8+ thymocytes for differentiation into SP T cells, which involves TCR signaling, was found to be impaired in CD45- mice. Impor- tantly, the present study demonstrates that engagement ofthe external domains of CD45 can also significantly affect im- a mature CD4+CD8+ thymocytes in that it enhances CD4- o0 associated Lck tyrosine kinase activity, inhibits TCR upreg- ulation, and inhibits the further differentiation of immature CD4+CD8+ thymocytes into mature SP T cells. In vivo anti-CD45 treatment does not alter CD45 activity as mea- sured directly by in vitro assays of phosphatase activity (data CD4 not shown). Rather, we think that in vivo anti-CD45 mAb FIG. 4. Treatment with mAb to CD45 inhibits the generation of treatment induces a redistribution of CD45 such that it CD8+ thymocytes in HY-specific TCR transgenic mice. Newborn specifically clusters with CD4, facilitating an interaction with H-2b female mice transgenic for the male-specific TCR were injected CD4-associated Lck molecules (30). As a consequence, daily from birth with saline or IgG anti-CD45 mAb. Three-color CD45 efficiently dephosphorylates the negative regulatory fluorescence analyses for CD4, CD8, and transgenic TCR idiotype tyrosine phosphorylation site (Tyr-505) on CD4-associated (T3.70) were performed on thymocytes from control females (Left) Lck molecules, increasing activity of CD4-associated Lck and mAb-treated female mice (Right) at 3 weeks of age. List-mode data collected on 250,000 viable cells (as determined by forward light (31). It is likely that engagement of external CD45 domains by scatter intensity and propidium iodide exclusion) were software potential ligands on thymic epithelial cells may similarly gated for T3.70+ cells, and data are displayed as contour diagrams of function to redistribute CD45 molecules on CD4+CD8+ thy- CD4 versus CD8 fluorescence intensity. Numbers within each box mocytes to localized patches in the plasma membrane that indicate the frequency of cells within that box. include CD4 and that contact class II+ thymic epithelial cells. In conclusion, the present study demonstrates that the Center and Right). However, the addition of anti-CD45 mAb ability of immature CD4+CD8+ thymocytes to undergo pos- to fetal thymic organ cultures markedly interfered with the itive selection can be regulated by engagement of CD45 generation of mature SP thymocytes (Fig. 3 Center). Indeed, external domains, suggesting an important regulatory role for anti-CD45-treated populations were devoid of intrathymic ligands that might be capable of engaging CD45 mature CD5hiTCRhi thymocytes (Fig. 3 Right), indicating that on CD4+CD8+ thymocytes. the reduced number ofSP thymocytes that were present were immature precursor cells rather than mature T cells. Finally, We are grateful to Drs. K. Katz and J. Punt for critically reading we examined the effect of anti-CD45 engagement on the the manuscript. P.B. is a postdoctoral fellow of the National Cancer positive selection of CD8+ T cells in mice expressing trans- Institute of Canada. D.L.W. is supported by a postdoctoral fellow- genic TCR specific for an antigenic complex composed of ship from the Cancer Research Institute, New York. H-2Db and the male HY antigen (23) (Fig. 4). Transgenic T cells are selected to become CD8+ T cells because of the 1. Singer, A., Mizuochi, T., Munitz, T. I. & Gress, R. E. (1986) major histocompatibility complex class I restriction specific- Prog. Immunol. 6, 60-66. TCR and can be identified by the 2. Fowlkes, B. J. & Pardoll, D. M. (1989) Adv. Immunol. 44, ity of the transgenic 207-264. (24) (Fig. 4 Left). However, neo- anti-idiotypic mAb T3.70 3. Ohashi, P. S., Pircher, H., Bfirki, K., Zinkernagel, R. M. & natal injection with anti-CD45 mAb inhibited the generation Hengartner, H. (1990) Nature (London) 346, 861-863. of mature T3.70+ CD8+ transgenic thymocytes (Fig. 4 Right), 4. Wiest, D. L., Yuan, L., Jefferson, J., Benveniste, P., Tsokos, demonstrating that CD45 engagement interfered with the M., Klausner, R. D., Glimcher, L., Samelson, L. E. & Singer, ability of immature transgenic thymocytes to undergo posi- A. (1993) J. Exp. Med. 178, 1701-1712. tive selection. 5. McCarthy, S. A., Kruisbeek, A. M., Uppenkamp, I. K., Shar- row, S. 0. & Singer, A. (1988) Nature (London) 336, 76-79. 6. Bonifacino, J., McCarthy, S. A., Maguire, J. E., Nakayama, DISCUSSION T., Singer, D. S., Klausner, R. D. & Singer, A. (1990) Nature The present study demonstrates that engagement of CD45 (London) 344, 247-251. June, C. H., Munitz, T. I., Sheard, M., Mc- on thy- 7. Nakayama, T., external domains expressed immature CD4+CD8+ Carthy, S. A., Sharrow, S. O., Samelson, L. E. & Singer, A. mocytes can regulate their further differentiation into mature (1990) Science 249, 1558-1561. SP T cells. The inhibitory effect of CD45 engagement on 8. Nakayama, T., Samelson, L. E., Nakayama, Y., Munitz, T. I., differentiation of CD4+CD8+ thymocytes parallels its en- Sheard, M., June, C. H. & Singer, A. (1991) Proc. Natl. Acad. hancement of CD4-associated Lck tyrosine kinase activity Sci. USA 88, 9949-9953. and its inhibition of TCR upregulation in CD4+CD8+ thymo- 9. Amrein, K. E. & Sefton, B. M. (1988) Proc. Nat!. Acad. Sci. cytes. Because Lck tyrosine kinase activity is known to be USA 85, 4247-4251. critical for thymocyte development (25-27), we think it likely 10. Mustelin, T., Coggeshall, K. M. & Altman, A. (1989) Proc. that the observed inhibition by anti-CD45 of thymocyte Nat!. Acad. Sci. USA 86, 6302-6306. differentiation is a consequence of the enhancing effect of 11. Ostergaard, H. L., Shackelford, D. A., Hurley, T. R., anti-CD45 on CD4-associated Lck tyrosine kinase activity. Johnson, P., Hyman, R., Sefton, B. M. & Trowbridge, I. S. However, a direct cause-and-effect relationship between the (1989) Proc. Nat!. Acad. Sci. USA 86, 8959-8963. effects of anti-CD45 on Lck activity and the effects of 12. Pingel, J. T. & Thomas, M. L. (1989) Cell 58, 1055-1065. 13. Desai, D. M., Sap, J., Schlessinger, J. & Weiss, A. (1993) Cell anti-CD45 on thymocyte development remains to be demon- 73, 541-554. strated. 14. Springer, T., Galfre, G., Secher, D. S. & Milstein, C. (1978) The results ofthis study complement, but are distinct from, Eur. J. Immunol. 8, 539-551. the recent observation that thymocyte differentiation was 15. Sarmiento, M., Glasebrook, A. L. & Fitch, F. W. (1980) J. arrested at the CD4+CD8+ stage of development in CD45- Immunol. 125, 2665-2672. mice whose CD45 gene was disrupted by homologous re- 16. Dialynas, D. P., Quan, Z. S., Wall, K. A., Pierres, A., Quin- combination (28). Since TCR signaling is known to be im- tans, J., Loken, M. R., Pierres, M. & Fitch, F. W. (1983) J. paired in CD45- cell lines (29), it is perhaps not so surprising Immunol. 131, 2445-2455. Downloaded by guest on October 2, 2021 Immunology: Benveniste et al. Proc. Nat!. Acad. Sci. USA 91 (1994) 6937

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