CD4 and CD8 Regulate Interleukin 2 Responses of T Cells

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5557-5561, June 1992 Immunology

CD4 and CD8 regulate interleukin 2 responses of T cells (Lyt-2/L3T4/p56I&/protein-tyrosine kinase) KAZUHISA TAKAHASHI*t, MOTOMI NAKATAt, TOSHIYUKI TANAKAt, HIROYASU ADACHIt, HIROMITSu NAKAUCHI§, HIDEO YAGITAt, AND Ko OKUMURAt¶ Departments of *Respiratory Medicine and tImmunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; tDepartment of Immunology, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan; and 9Laboratory of Molecular Regulation of Aging, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Yatabe, Tsukuba, Ibaraki 305, Japan Communicated by Leonard A. Herzenberg, March 26, 1992

ABSTRACT To characterize the T-cell surface molecules MATERIALS AND METHODS involved in regulation of T-cell interleukin 2 (IL-2) responses, we established several monoclonal antibodies (mAbs) that Cell Lines. Murine IL-2-dependent T-cell lines CTLL-2 inhibit IL-2 responses of freshly isolated CD8+ T cells and the (18) and HT-2 (19) (kindly provided by H. Karasuyama, Tokyo University) were maintained in RPMI 1640 (Nissui IL-2-dependent cell line CTLL-2. Here we show that two Pharmaceutical, Tokyo) supplemented with 10% heat- mAbs are directed against Lyt-2 (CD8a). In fact, all inhibitory inactivated fetal bovine serum (Bocknek Laboratories, anti-Lyt-2 mAbs tested were able to inhibit the IL-2 response Rexdale, ON, Canada), penicillin (100 units/ml), streptomy- of the Lyt-2- and L3T4-dericient cell line HT-2 after transfec- cin sulfate (50 ,g/ml), and 2 mM L-glutamine (complete tion with a Lyt-2 cDNA clone. Similarly, anti-L3T4 mAbs medium) containing recombinant human IL-2 (100 units/ml; inhibited the IL-2 response of CD4-transfected HT-2 cells. Shionogi Pharmaceutical, Osaka). P815 murine mastocytoma These inhibitory effects of anti-CD4 and anti-CD8 mAbs occur cells (American Type Culture Collection) were also main- on normal T lymphocytes, since they also were observed with tained in complete medium. CD4+ and CD8+ T-cell blasts, and are specific for IL-2 Antibodies. PC61 (anti-murine IL-2Ra) (20), 53-6.72 (anti- responses, since IL-4 responses of CD4- and CD8-transfected murine CD8a or anti-Lyt-2) (21), GK1.5 (anti-murine CD4 or HT-2 cells were not affected by the anti-CD4 and anti-CD8 anti-L3T4) (22), and 145-2C11 (anti-CD3) (23) mAbs were mAbs. The inhibitory effects of anti-CD4 or anti-CD8 mAbs provided by H. R. MacDonald (Ludwig Institute), L. A. could not be explained by interference with IL-2 binding and Herzenberg (Stanford University), F. W. Fitch (Chicago depended on CD4 and CD8 crosslinking, because F(ab')2 or University), and J. A. Bluestone (Chicago University), re- Fab plus crosslinking second antibody, but not Fab alone, were spectively. Anti-Lyt-2.2 and anti-Lyt-2.1 (allotypic anti- effective. A mutant Lyt-2 molecule lacking the cytoplasmic Lyt-2 mAbs) were gifts from Meiji Milk Product (Tokyo). region that mediates p56kk binding could not mediate the Anti-Lyt-3.2 and anti-Lyt-3.1 (HO-3.1) (allotypic anti-Lyt-3 inhibitory effect upon crosslinking. These results suggest that mAbs) were kindly provided by E. Nakayama (Nagasaki CD4 and CD8 mediate negative regulation of T-cell IL-2 University). F(ab')2 and Fab fragments of the mAbs were responses via cytoplasmically associated p56kk. prepared as described (24). Immunizations and Production of mAbs. Adult SD rats Interleukin 2 (IL-2) plays a central role in regulating immune (Sankyo Labo Service, Tokyo) were primed with an intra- responses (1). IL-2-induced activation and differentiation dermal injection of 107 T-cell blasts [prepared by culturing signals are mediated by specific receptors on the cell surface. BALB/c spleen cells with Con A (5 ,ug/ml) for 2 days] in The IL-2 receptor (IL-2R) is composed of two polypeptides, complete Freund's adjuvant, followed by a booster injection, p55 IL-2Ra and p75 IL-2RB. p55 or p75 alone constitute low- 10 days later, containing the same number of T-cell blasts in or intermediate-affinity receptor, respectively, but only the incomplete Freund's adjuvant, and i.p. injection of T-cell latter is functional in mediating signals. The combination of blasts in phosphate-buffered saline three times per week. both subunits constitutes the functional high-affinity IL-2R After 10 days, antisera were collected and antibody activities (2-6). cDNA transfection studies revealed that p55 and p75 against T-cell blasts were estimated by immunofluorescence. IL- Animals developing a high titer of antibody were given a final were sufficient for reconstituting functional high-affinity of T-cell blasts (2 x Immune splenocytes not on i.p. injection 107). 2Rs on hematopoietic cell lines, but nonhematopoietic were prepared 3 days later and fused with P3X63Ag8-653 cell lines (7-13). Similarly, p75 alone reconstituted the func- murine myeloma cells (25). We screened for antibodies that tional intermediate-affinity receptor only on hematopoietic inhibited IL-2 induction of anti-CD3-redirected cytotoxic cell lines (7, 12). These results suggest that other hemato- activity in mouse spleen T cells as described below. mAbs poietic components in addition to p55 and p75 are required for 1C7-3, 2F4, 5D7-1, 5D9-2, and 5H10-1 were selected for their constitution of the functional IL-2R. In this respect, candi- strong inhibitory effects and cloned by limiting dilution. dates for IL-2R-associated molecules have been reported Cytotoxicity Assay. Spleen cells from BALB/c mice were (14-17). To further investigate the molecules involved in cultured with recombinant human IL-2 (1000 units/ml) in the regulating IL-2 responses, we established several monoclo- presence of the indicated mAb (10 ,ug/ml) with or without nal antibodies (mAbs) that inhibit T-cell IL-2 responses, two PC61 (10 ,ug/ml). After 2 days, the cells were harvested and of which reacted with Lyt-2. Here we describe the involve- their cytotoxic activity against Fc,, receptor-bearing P815 ment of CD4, CD8, and their association with p56lck in cells in the presence of anti-CD3 mAb (0.1 ,ug/ml) was tested regulating IL-2 responses of T cells. in a standard 4-hr 5lCr-release assay. The effector/target cell

The publication costs of this article were defrayed in part by page charge Abbreviations: IL, interleukin; IL-2R, IL-2 receptor; mAb, mono- payment. This article must therefore be hereby marked "advertisement" clonal antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1To whom reprint requests should be addressed.

5557 Downloaded by guest on October 2, 2021 5558 Immunology: Takahashi et al. Proc. Natl. Acad Sci. USA 89 (1992) ratio was 50:1, and percent cytotoxicity was calculated as molecule immunoprecipitated by an anti-Lyt-2 mAb, 53-6.72. described (26). These appeared to be identical. In sequential immunoprecip- Proliferative Response of IL-2- and IL-4-Dependent Cell itation studies, preclearing of CD8 from a thymocyte lysate Lines. IL-2/IL-4-dependent cell lines were first deprived of with the anti-Lyt-2 mAb completely depleted the antigen IL-2 for >2 hr in complete medium. They were cultured at reactive with 5D9-2 and 5H10-1, and vice versa (data not 370C for 24 hr in complete medium containing serially diluted shown). In addition, 5D9-2 and 5H10-1 competed with fluo- IL-2 or IL-4 in the presence or absence of the mAbs (10 rescein-labeled 53-6.72 for binding to thymocytes as esti- ,zg/ml) with or without PC61 (10 ,ug/ml). Proliferation was mated by flow cytometry. Based on these data, we concluded measured by incorporation of [3H]thymidine (1 ,tCi per well; that 5D9-2 and 5H10-1, which inhibited IL-2 responses, are 1 Ci = 37 GBq) during the last 4 hr of the culture. directed to the Lyt-2 (CD8a) molecule. To determine whether Proliferative responses of CD81 or CD4+ T-cell blasts, the inhibitory effect on IL-2 response is unique for 5H9-2 and which were induced from CD8+ and CD4+ spleen cells by 5H10-1, or a general property of anti-CD8 mAbs, we used a culturing with phorbol 12-myristate 13-acetate (10 ng/ml), panel of anti-Lyt-2 and anti-Lyt-3 (CD8f3) mAbs. All anti- ionomycin (500 ng/ml), and recombinant human IL-2 (50 Lyt-2 mAbs tested significantly inhibited the IL-2 response of units/ml) for 5 days, were tested in a similar way. CTLL-2, while this effect was not observed with irrelevant Immunoprecipitation. Surface radioiodination of BALB/c allotypic anti-Lyt-2.1 and anti-Lyt-3.1 mAbs (Fig. 1). An thymocytes and immunoprecipitation were performed as anti-Lyt-3.2 mAb did react with CTLL-2 to a similar extent described (27). as an anti-Lyt-2.2 as CD4 and CD8 Expression Vectors and Transfection. The mAb, estimated by flow cytometry, but expression constructs containing of the full-length wild-type it did not inhibit the IL-2 response. These results indicate that Lyt-2 cDNA or the mutant cDNA whose cytoplasmic region the inhibitory effect on the IL-2 response is a general prop- was truncated (Lyt-2-107A4) were prepared as described (28). erty of anti-Lyt-2 mAbs but not of anti-Lyt-3 mAbs. The expression construct containing the full-length L3T4 Inhibitory Effects of Anti-Lyt-2 and Anti-L3T4 mAbs on (pSFSVn-LC7) (29) was kindly provided by J. Parnes (Stan- IL-2 and IL-4 Responses of CD4- or CDga-Transfected HT-2 ford University). HT-2 cells were transfected with those Cells. To further clarify the inhibitory effect of anti-Lyt-2 constructs by electroporation (30). Selection with G418 (1 mAb on IL-2 responses, we introduced Lyt-2 into an IL-2- mg/ml) was started 48 hr after transfection. G418-resistant dependent cell line, HT-2, by cDNA transfection. HT-2 cells expressing a high level of Lyt-2 or L3T4 on the surface originally did not express CD8 or CD4 as determined by flow were isolated by fluorescence-activated cell sorting. cytometry and Northern blot analysis (data not shown). HT-2 IL-2 Binding Assay. IL-2 binding and Scatchard analysis transfectants expressing high levels ofLyt-2 were isolated by were done as described (11). fluorescence-activated cell sorting, and the effect of anti- Immunoprecipitation and Immunoblotting with Anti-p56ck Lyt-2 mAbs on the IL-2 response was examined (Fig. 2). IL-2 Antibody. These procedures were performed as described response ofthe CD8a (Lyt-2) transfectant, but not that ofthe (28). original HT-2 cells, was inhibited by 53-6.72 in a dose- dependent manner. Similar effects were observed with 5D9-2 and 5H10-1 (data not shown). We also established a CD4 RESULTS (L3T4)-transfected HT-2 cell line, and observed that its IL-2 Establishment and Characterization of mAbs That Inhibit response was inhibited to a higher extent by an anti-CD4 mAb IL-2 Responses of Murine T Cells. We first tried to establish (GK1.5) (Fig. 2), indicating that CD4 is also involved in a mAb inhibiting IL-2 response of an IL-2-dependent cell line, HT-2, but the resulting mAbs were found to be directed W11,111, to p55 IL-2Ra, probably due to its extremely high expression on HT-2. Based on our previous observation in human r fJ-2_ systems that cytotoxic activation ofperipheral blood CD8+ T cells is mediated solely by p75 IL-2Rj (31), we next screened for mAbs inhibiting the induction by IL-2 of cytotoxic activity of mouse splenic T cells in an anti-CD3-redirected -- ;.-`. 2 cytotoxicity assay. We selected five mAbs (1C7-3, 2F4, 5D7-1, 5D9-2, 5H10-1) that did not inhibit the anti-CD3- redirected cytotoxicity significantly; control anti-Thy-1 mAb exhibited no inhibitory effect, excluding the possibility that the mAbs simply compete with anti-CD3 mAb for Fc receptor P-ser VillIllI"a0PA'villaP.,"m binding to the target cells (data not shown). In addition, these mAbs alone could partially inhibit the IL-2 response of the EVANNIFFAIVINPA'0VlolPA,VA0. IL-2-dependent cell line CTLL-2, as well as cytotoxic induction of splenic T cells, and acted synergistically with an anti-p55 IL-2Ra mAb (PC61) in both systems (data not shown). Among these five mAbs, two (5D9-2 and 5H10-1) reacted weakly with CTLL-2 and very strongly with the FIG. 1. Inhibitory effects of anti-CD8a (Lyt-2) and anti-CD83 majority of thymocytes and a subset of spleen cells as (Lyt-3) mAbs on IL-2-induced proliferation of CTLL-2 cells. 5D9-2, estimated by indirect immunofluorescence and flow cytom- 5H10-1, and 53-6.72 are rat mAbs against Lyt-2. Lyt-2.2 and Lyt-3.2 etry. The antigen molecules on thymocytes recognized by are relevant allotypic epitopes expressed on CTLL-2 cells. Lyt-2.1 these two mAbs were two disulfide-linked heterodimers and Lyt-3.1 are irrelevant allotypic epitopes not expressed on composed of 38- and 30-kDa subunits or 35- and 30-kDa CTLL-2 cells. CTLL-2 cells (104) in 200 jl ofcomplete medium were subunits, as estimated by immunoprecipitation and SDS/ cultured for 24 hr with recombinant human IL-2 (100 units/ml) in the presence of the indicated mAbs (each at 10 ,tg/ml) with (filled bars) PAGE (data not shown). The distribution and biochemical or without (hatched bars) PC61 anti-IL-2Ra mAb (10 ug/ml). Cell characteristics of the antigen recognized by 5D9-2 and proliferation was measured by [3H]thymidine incorporation during 5H10-1 were reminiscent of murine CD8. Therefore, we the last 4 hr of the culture. Data represent mean + SD of triplicate compared the apparent molecular mass of the antigen immu- samples. Dotted line indicates the [3H]thymidine incorporation in the noprecipitated by 5D9-2 and 5H10-1 with that of the CD8 absence of IL-2. Downloaded by guest on October 2, 2021 Immunology: Takahashi et al. Proc. NatL. Acad. Sci. USA 89 (1992) 5559

Z ~~~~~~~~4 5- 0 2 x E 0~~~~~~~~ 0. (-) Intact F(ab')2 Fab Fab+ag (-) Intact F(ab')2 Fab Fab+ag 0 Anti-Lyt-2 Anti-L3T4 In FIG. 3. Requirement for crosslinking of Lyt-2 and L3T4 by anti-Lyt-2 and anti-L3T4 mAbs to inhibit IL-2 response. HT-2 Lyt-2 HT-2 L3T4 and HT-2 L3T4 transfectants were cultured with recombinant IL-2 (100 units/ml) in the presence or absence of anti-Lyt-2 (53-6.72) or 20 anti-L3T4 (GK1.5) mAb, respectively. Each antibody or antibody fragment [F(ab')2 or Fab] was added (10 ,Lg/ml). The monovalent Fab fragments were tested with and without anti-rat Ig (alg). IL-2 10 response was measured as in Fig. 1. Data represent mean + SD of triplicate samples. t% M61r-- 0 wv w 0.01 0.1 1 10 100 1000 0.01 0.1 1 10 100 ments were inhibitory only after crosslinking by anti-rat IL-2, units/ml IL-4, units/ml immunoglobulin (Fig. 3). FIG. 2. Effects of anti-Lyt-2 and anti-L3T4 mAbs on IL-2 and To determine whether the inhibitory effects of anti-CD8 IL-4 responses of HT-2, Lyt-2-transfected HT-2 (HT-2 Lyt-2), and and anti-CD4 mAbs on IL-2 responses of IL-2-dependent L3T4-transfected HT-2 (HT-2 L3T4). The indicated cells (104) in T-cell clones could be generated on normal T cells, we complete medium were cultured for 24 hr with serially diluted prepared CD8+ and CD4+ T-cell blasts from spleen cells by recombinant IL-2 (Left) or IL-4 (Right) in the absence ofantibody (o) isolating each population by fluorescence-activated cell sort- or in the presence (10 ,ug/ml) ofanti-Lyt-2 (53-6.72) (o) or anti-L3T4 ing and activated them with phorbol ester and ionomycin. (GK1.5) (A) mAb. Cell proliferation was measured as in Fig. 1. Data Anti-Lyt-2 and anti-L3T4 mAbs also significantly inhibited represent mean + SD of triplicate samples. the IL-2 response of CD8+ and CD4+ T cell blasts, respec- regulation of IL-2 responses. In contrast, control anti-Thy-1 tively (Fig. 4). These results suggest that crosslinking of CD4 mAb did not inhibit the IL-2 responses of these transfectants as well as CD8 leads to a specific down-regulation of IL-2 (data not shown). responses. In addition to IL-2, HT-2 can also respond to IL4. In Involvement of Lyt-2-Associated p56Ick in Negative Regula- contrast to the IL-2 response, however, the IL-4 response of tion of IL-2 Responses. Anti-Lyt-2 and anti-L3T4 mAbs used the CD8a- or CD4-transfected HT-2 was not affected by above did not affect the binding of IL-2 to the CD8a- or anti-Lyt-2 or anti-L3T4 mAbs (Fig. 2), indicating that the CD4-transfected HT-2 cells as estimated by Scatchard anal- inhibitory effects were specific for IL-2 responses. ysis (data not shown). Therefore, inhibitory effects of these The inhibitory effects ofanti-Lyt-2 and anti-L3T4 mAbs on mAbs on IL-2 responses were likely to be directed to the IL-2 response of the CD8a- or CD4-transfected HT-2 cells signal-transduction pathway following binding of IL-2 to its were dependent on the extent of CD8 or CD4 crosslinking. receptor. IL-2R-mediated signal transduction has been re- More extensive crosslinking using anti-rat immunoglobulin ported to involve tyrosine phosphorylation (32-35), while augmented the inhibitory effects of anti-Lyt-2 and anti-L3T4 CD4- and CD8-mediated signals have been supposed to be mAbs (data not shown). Divalent F(ab')2 fragments were mediated by the protein-tyrosine kinase p561ck, which asso- equally effective as intact mAbs, but monovalent Fab frag- ciates with the cytoplasmic portion of these molecules (36-

x E

0 :I-

0.01 0.1 1 10 100 1000 0.01 0.1 10 100 1000 IL-2, units/ml

FIG. 4. Effect of anti-Lyt-2 and anti-L3T4 mAbs on IL-2 responses of CD8+ or CD4+ T-cell blasts. CD8+ and CD4+ splenic T cells were isolated by fluorescence-activated cell sorting and cultured with phorbol 12-myristate 13-acetate (10 ng/ml), ionomycin (500 ng/ml), 4nd recombinant IL-2 (50 units/ml) for 5 days. The T-cell blasts were then cultured with serially diluted IL-2 in the presence (e) or absence (o) of anti-Lyt-2 (53-6.72) or anti-L3T4 (GK1.5) mAb at 10 ,ug/ml. Cell proliferation was measured as in Fig. 1. Data represent mean ± SD of the triplicate samples. Downloaded by guest on October 2, 2021 5560 Immunology: Takahashi et al. Proc. Natl. Acad Sci. USA 89 (1992)

HT-2 Lyt-2 HT-2 Lyt-2 -107i 4 HT-2 L-3 I-a, involved in regulation of IL-2 responses, we established 4.D)- several mAbs that inhibit IL-2 responses of murine T cells. Unexpectedly, some of them were directed to the CD8a (Lyt-2) molecule. In addition, all anti-Lyt-2 and anti-L3T4 .- mAbs tested could inhibit IL-2 responses of splenic T-cell _ I- Al" _N.Po blasts, as well as those of the IL-2-dependent cell line p56' ~ CTLL-2 and CD8- or CD4-transfected HT-2 cells. However, these mAbs could not inhibit IL-4 responses of CD4- or CD8-transfected HT-2 cells, indicating that the inhibitory 33 effects on IL-2 responses are not due to nonspecific inhibition ofcell growth. Moreover, anti-Lyt-2 and anti-L3T4 mAbs did 1 2 3 4 5 6 not affect the binding of IL-2 to Lyt-2- or L3T4-transfected FIG. 5. Expression of p5ock and its interaction with Lyt-2 and HT-2 cells, suggesting that the inhibitory effect involves the L3T4 in HT-2 transfectants. Immunoprecipitates were examined by signal transduction cascade via IL-2R. immunoblotting with anti-p56ock antibody. Lanes 1 and 2, HT-2 IL-2 stimulation can lead to tyrosine phosphorylation of Lyt-2; lanes 3 and 4, HT-2 Lyt-2-107A4; lanes 5 and 6, HT-2 L3T4 several substrates, including the p75 IL-2Rf3 chain itself transfectants. Lanes 1, 3, and 5 represent one-fifth of anti-p56ock (32-35). In addition, IL-2 responses can be inhibited by immunoprecipitates; lanes 2 and 4 represent anti-Lyt-2 (53-6.72) protein-tyrosine kinase inhibitors (39). These reports suggest immunoprecipitates; and lane 6 represents anti-L3T4 (GK1.5) im- that protein-tyrosine kinases might be involved in the signal- munoprecipitates. transduction cascade via IL-2R, especially since neither p75 38). Therefore, it was feasible to test the involvement of nor p55 has a tyrosine kinase domain (7). Hatakeyama et al. We the (40) reported that a lymphocyte-specific tyrosine kinase, p56lck in the inhibitory mechanisms. introduced p561ck, is physically associated with p75 IL-2Rf3 and that its cDNA encoding a mutant Lyt-2 molecule (Lyt-2-107A4) kinase activity was promoted by IL-2 binding. Horak et al. whose cytoplasmic portion was replaced with 8 nonsense (41) reported a similar increase of p561ck kinase activity after amino acids instead of28 amino acid residues ofthe wild-type IL-2 stimulation ofIL-2-dependent T-cell lines. These results Lyt-2 and, therefore, is unable to bind to p56lck (28). The suggest that p561ck associated with p75 may have a role in mutant Lyt-2 molecule was expressed on HT-2 cells at a level regulating IL-2R-mediated signal transduction. On the other similar to that of the transfected wild-type Lyt-2 (data not hand, p56ck was first demonstrated to be associated with the shown). The wild-type, but not the mutant, Lyt-2 coprecip- cytoplasmic domains of CD8a and CD4 (36-38). CD4 or CD8 itated p56Ick as shown by immunoblotting of the anti-Lyt-2 crosslinking by anti-CD4 or anti-CD8 mAb promoted the immunoprecipitates with anti-p561ck antibody (Fig. 5). L3T4 kinase activity only of p561ck that associated with CD4 or coprecipitated more p56Ick than wild-type Lyt-2, as previ- CD8. Therefore, our present observation that CD4 or CD8 ously reported (37). By densitometry, 30%, 10%1, and <0.1% crosslinking down-regulates IL-2 responses via associated of total cellular p56Ick was associated with L3T4, wild-type p5o6ck suggests a direct physical linkage between p75 IL-2Rf Lyt-2, and mutant Lyt-2, respectively, in each transfectant. and CD4 or CD8a via the same p56ock molecule, although it In contrast to the wild-type Lyt-2 transfectant, IL-2 as well is also possible that the inhibition may reflect an indirect as IL-4 responses of the mutant Lyt-2 transfectant were not mechanism, following some signal-transduction event result- inhibited by anti-Lyt-2 mAb (Fig. 6). Therefore, the inhibi- ing from crosslinking CD4 or CD8. This notion is consistent tory effect of CD4 and CD8 crosslinking on IL-2 responses with the fact that CD8a or CD4 and p75 IL-2Rf bind to appears to be correlated strongly with the amount of p56lck distinct sites on p56lck (40). Further biochemical studies will associated with these molecules, suggesting the involvement be needed to test this possibility. However, p561ck is not ofp56Ick in the negative regulation ofIL-2 responses resulting always required for IL-2R-mediated signal transduction, be- from CD4 and CD8 crosslinking. cause the growth of some cell lines lacking p5ock can be promoted by IL-2 after introduction of p75 (11, 40). More- DISCUSSION over, a deletion mutant of p75 that cannot bind p561ck can work as a functional receptor (7, 40). Therefore, p561ck Molecular cloning of p55 and p75 components of the IL-2R appears to be not obligatory but regulatory for IL-2 re- has enabled reconstitution studies to be performed by cDNA sponses, as demonstrated by our present study. transfection (7-13). They revealed that molecules other than Crosslinking of CD4 by anti-CD4 mAb prior to T-cell p55 and p75 may be required for signal transduction via receptor crosslinking can lead to T-cell unresponsiveness due IL-2R. Several candidate molecules associating with the to the induction of apoptosis (42), and in vivo administration IL-2R have been reported (14-17). However, their functional of anti-CD4 mAb can lead to allograft tolerance by inducing properties have not been clarified. To characterize molecules clonal anergy (43). Although the molecular mechanisms for

FIG. 6. Inhibitory effects of 0 anti-Lyt-2 mAb on IL-2 and IL-4 x responses of HT-2 Lyt-2-107A4 E transfectants. The cells were cul- 0 tured with serially diluted recombinant IL-2 (Left) or IL-4 (Right) in the presence (-) or absence (o) of anti-Lyt-2 (53-6.72) mAb at 10 Ag/ml for 24 hr. Cell proliferation was measured as in Fig. 1. Data 1 100 represent mean + SD of triplicate IL-2, units/ml IL-4, units/ml samples. Downloaded by guest on October 2, 2021 Immunology: Takahashi et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5561 these phenomena have not been clarified, down-regulation of 8. Hatakeyama, M., Mori, H., Doi, T. & Taniguchi, T. (1989) Cell 59, by CD4 crosslinking might be respon- 837-845. IL-2 responsiveness 9. Tsudo, M., Karasuyama, H., Kitamura, F., Nagasaka, Y., Tanaka, sible for the induction of apoptosis and clonal anergy. T. & Miyasaka, M. (1989) J. Immunol. 143, 4039 4043. The physiological consequences of our present observa- 10. Doi, T., Hatakeyama, M., Minamoto, S., Kono, T., Mori, H. & tions remain to be determined. CD4 and CD8 can bind Taniguchi, T. (1989) Eur. J. Immunol. 19, 2375-2378. class II or class I major histocompatibility com- 11. Tanaka, T., Tsudo, M., Karasuyama, H., Toyama, N., directly to Hatakeyama, M., Taniguchi, T. & Miyasaka, M. (1991) Int. Immu- plex molecules, respectively, independently of the T-cell nol. 3, 105-108. receptor (44). Although their binding affinity appears to be so 12. Tsudo, M., Karasuyama, H., Kitamura, F., Tanaka, T., Kubo, S., weak that the binding between CD4+ cells and class II- Yamamura, Y., Tamatani, T., Hatakeyama, M., Taniguchi, T. & cells could be observed only when these molecules Miyasaka, M. (1990) J. Immunol. 145, 599-606. positive 13. Okamoto, Y., Minamoto, S., Shimizu, K., Mogami, H. & Tanigu- were greatly overexpressed, such a weak interaction would chi, T. (1990) Proc. Nadl. Acad. Sci. 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Immunol. 18, 97-104. stander activation would be particularly needed to direct 20. Lowenthal, J. W., Corth6sy, P., Tougne, C., Lees, R., MacDonald, CD8+ T cells to antigen-specific activation, since they con- H. R. & Nabholz, M. (1985) J. Immunol. 135, 3988-3994. stitutively express p75 IL-2R( and can respond to IL-2 21. Ledbetter, J. A. & Herzenberg, L. A. (1979) Immunol. Rev. 47, 63-90. without antigen stimulation (31). 22. Wilde, D. B., Marrack, P., Kappler, J., Dialynas, D. P. & Fitch, Another possibility is suggested by the observation that F. W. (1983) J. Immunol. 131, 2178-2183. CD4 or CD8 crosslinking down-regulates IL-2 but not IL-4 23. Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. & Bluestone, responsiveness. It was reported that IL-4 inhibited the IL-2 J. A. (1987) Proc. Nati. Acad. Sci. USA 84, 1374-1378. 24. Nitta, T., Yagita, H., Azuma, T., Sato, K. & Okumura, K. (1989) induction of lymphokine-activated killer (LAK) activity but Eur. J. 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