Hyporesponsiveness in Nonobese Diabetic Mice TCR Complex May ...

Sequestration of CD4-Associated Lck from the TCR Complex May Elicit T Cell Hyporesponsiveness in Nonobese Diabetic Mice1 Jian Zhang,2* Konstantin Salojin,2* and Terry L. Delovitch3*† The Lck protein tyrosine kinase associates noncovalently with the cytoplasmic domain of CD4. Upon ligand engagement of the TCR, CD4-associated Lck is rapidly activated and recruited to the TCR complex. Coupling of this complex to an intracellular signaling pathway may result in T cell proliferation. Previously, we reported that thymocytes from nonobese diabetic (NOD) mice (>6 wk of age) exhibit a proliferative hyporesponsiveness after TCR stimulation, which is associated with defective TCR-mediated signaling along the protein kinase C/Ras/mitogen-activated protein kinase pathway of T cell activation. Here, we investigated whether differential association of Lck with TCR or CD4 mediates the control of NOD thymocyte hyporesponsiveness. We demonstrate that less CD4-associated Lck is recruited to the TCR in activated NOD thymocytes than in control thymocytes. This CD4-mediated sequestration of Lck from the TCR correlates with the increased binding of CD4-associated Lck through its Src homology 2 domain to free TCRz and CD3ge chains on the plasma membrane. Sequestration of Lck by CD4 does not occur in activated thymocytes from 3-wk-old NOD mice and is only apparent in thymocytes from NOD mice >5 to 6 wk of age. This diminished recruitment of CD4-associated Lck to the TCR is not mediated by an increase in the amount of CD8-associated Lck. Thus, impaired recruitment of CD4-associated Lck to the TCR complex may represent an early event that results in deficient coupling of the TCR complex to downstream signaling events and gives rise to NOD thymocyte hyporesponsiveness. The Journal of Immunology, 1998, 160: 1148 –1157.

T

he TCR is a multimolecular complex consisting of at least six different transmembrane proteins derived from three protein families: clonotypic a- and b-chains that are expressed as disulfide-linked heterodimers and determine the recognition specificity of the complex; the invariant g-, d-, and e-chains of the CD3 complex that noncovalently associate to form CD3ge and CD3de heterodimers; and z- and h-chains that exist either as disulfide-linked z-z homodimers or z-h heterodimers (1). The CD3 components and TCRz chains mediate the earliest TCR-transduced signals. Engagement of the TCR by Ag or by an anti-TCR mAb triggers a signal transduction cascade that induces various T cell responses (2, 3). One of the earliest detectable signaling events after TCR ligation is the tyrosine phosphorylation of a number of intracellular protein substrates, including Lck, a member of the src family protein tyrosine kinases (PTKs)4 (4 –7). In T cells, Lck binds noncovalently to the cytoplasmic domains of the CD4 and

*Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, and †Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada Received for publication June 26, 1997. Accepted for publication October 16, 1997. 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 a grant from the Juvenile Diabetes Foundation International, the Helen M. Armstrong grant from the Canadian Diabetes Association (to T.L.D.), Juvenile Diabetes Foundation International postdoctoral fellowships (to J.Z. and K.S.). 2

J.Z. and K.S. contributed equally to this work.

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Address correspondence and reprint requests to Dr. Terry L. Delovitch, Director, Autoimmunity/Diabetes Group, The John P. Robarts Research Institute, 1400 Western Rd., London, Ontario, Canada N6G 2V4. E-mail address: [email protected] 4 Abbreviations used in this paper: PTK, protein tyrosine kinase; NOD, nonobese diabetic; NOR, nonobese diabetes resistant; MAPK, mitogen-activated protein kinase; SH2/3, Src homology 2/3; B6, C57BL/6J; POD, peroxidase; GST, glutathione-S-transferase.

Copyright © 1998 by The American Association of Immunologists

CD8 coreceptors (8, 9). Ab-mediated cross-linking of CD4 or CD8 activates Lck (9 –12), and mutations of the CD4 or CD8 intracellular domains that disrupt their association with Lck prevent their capacity to enhance TCR-mediated activation (13). The deficient expression of Lck in T cells results in defective TCR-mediated signaling, which may be reconstituted by expression of the wildtype lck gene (5). Mice lacking a functional Lck gene (14) or overexpressing a catalytically inactive form of Lck (15) manifest an early arrest of thymocyte maturation. These findings indicate that Lck is essential for both T cell activation and T cell development. In a coreceptor model of T cell activation, the association of Lck with CD4 or CD8 is required for optimal T cell activation by Ag and for the effective selection of mature T cells during thymopoiesis (16 –21). The interaction of CD4 and CD8 with MHC class II and class I molecules, respectively, is thought to recruit the CD4and CD8-associated Lck complexes within the close proximity of ligand-occupied TCR/CD3 complexes. This recruitment initiates an intracellular signaling cascade mediated by the tyrosine phosphorylation of several substrates associated with the TCR complex. Consistent with this model, Ab co-cross-linking of CD4 with the TCR complex is more efficient in inducing tyrosine phosphorylation of TCR-associated proteins and T cell activation than cross-linking of the TCR complex alone (21–23). However, CD4 may also deliver a negative signal for T cell activation (24 –27). In the absence of Ag, the association of Lck with CD4 prohibits the induction of T cell growth signals through TCRab (24). Thus, optimal TCR-dependent signaling seems to require not only activation of Lck but also juxtaposition of Lck to the TCR complex (26). Previously, we reported that thymic and peripheral T cells in autoimmune nonobese diabetic (NOD) mice exhibit a proliferative hyporesponsiveness in vitro after TCR stimulation (28). 0022-1767/98/$02.00

The Journal of Immunology Exogenous IL-4, a Th2-type cytokine, potentiates IL-2 production, completely restores NOD T cell proliferative responsiveness in vitro, and prevents the onset of autoimmune diabetes in NOD mice in vivo. These findings suggest that IL-4-producing Th2-type cells may have a role in protection against the onset of diabetes in NOD mice (29). We also showed that this NOD T cell hyporesponsiveness is associated with defective TCR-mediated signal transduction along the protein kinase C/Ras/ MAPK pathway of T cell activation (30). Ras activation is deficient in quiescent and stimulated NOD T cells, and this correlates closely with the reduced activity of MAPK (30). This idea that T cell hyporesponsiveness is mediated by a block in Ras activation is further supported by the demonstration that altered Ras and MAPK/JNK kinase activities arise in anergic murine T cell clones (31, 32). More recently, we found that TCR-stimulated NOD thymocytes exhibit constitutive downregulation of Ras-associated GDP-releasing activity as a result of the inability of the mSOS guanine nucleotide-releasing factor to be translocated from the cytoplasm to the plasma membrane in association with Grb2 (33). However, it remains to be determined how TCR-proximal signaling events mediate these Rasassociated signaling defects and NOD T cell hyporesponsiveness. Although CD4 can block the TCR-induced growth of T cells, the mechanism of this growth inhibition is not understood. While this inhibition has been proposed to occur via the physical and/or functional sequestration of Lck by CD4 from the TCR (24, 27), direct biochemical evidence for this type of TCR-induced Lck sequestration has not yet been reported. Accordingly, we investigated whether Lck sequestration by CD4 mediates TCR-induced NOD thymocyte hyporesponsiveness. We demonstrate that rapidly after TCR stimulation of NOD thymocytes, CD4-associated Lck is sequestered from the TCR/CD3 complex, possibly by the binding of its SH2 domain to apparently free TCRz and CD3ge chains on the plasma membrane. Our data provide the first biochemical evidence for TCR-induced sequestration of Lck from the TCR by CD4. Further, they suggest that this impaired recruitment of CD4associated Lck to the TCR may be a relatively early event that elicits deficient coupling of the TCR to downstream signaling events and culminates in NOD T cell hyporesponsiveness.

Materials and Methods Mice NOD/Del and NOR/Lt mice were bred and maintained in our specific pathogen-free animal facility at The John P. Robarts Research Institute. C57BL/6J (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the Animal Care Facility at the University of Western Ontario (London, Canada). Female mice were used at 3 to 10 wk of age.

Reagents and Abs The 145-2C11 anti-CD3e mAb was supplied by Dr. J. Bluestone (University of Chicago, Chicago, IL). Biotinylated anti-CD4 (RM4-5), biotinylated anti-TCRb (H57-597), purified anti-TCRb (H57-597), and anti-CD4 mAb (RM4-4) were purchased from PharMingen (San Diego, CA). Anti-Lck mAb, anti-phosphotyrosine mAb (PY-20), and anti-TCRb (A-19) polyclonal Abs were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-Lck polyclonal Abs were provided by Dr. A. Veillette (McGill University, Montreal, Canada). Rabbit antisera 387 and 551 to TCRz chains were provided by Drs. L. E. Samelson and A. Singer, respectively (National Institutes of Health, Bethesda, MD). Rabbit antisera to CD3g and CD3d chains were provided by Drs. D. R. Alexander (University of Oxford, Oxford, U.K.) and L. E. Samelson, respectively. Lck SH2 and SH2/SH3 domain fusion proteins and mouse anti-Fyn mAb were obtained from Santa Cruz Biotechnology. The rabbit anti-hamster affinitypurified Ig, peroxidase (POD)-conjugated rabbit anti-goat IgG and ExAvidin-POD were purchased from Sigma Chemical Co. (Mississauga, Canada). Horseradish peroxidase-conjugated goat anti-mouse IgG and donkey anti-rabbit IgG were purchased from Amersham (Oakville, Canada).

1149 Anti-rat Ig-POD was obtained from Boehringer Mannheim Canada (Laval, Canada).

Thymocyte isolation, activation, and lysis NOD, B6, and NOR mice were killed, thymii were removed, and thymocyte single cell suspensions were prepared as previously described (29). Freshly isolated thymocytes were maintained on ice in DMEM supplemented with 20 mM HEPES (Life Technologies, Burlington, Canada) until use. For detection of the amount and activity of CD4-associated Lck, thymocytes were isolated at 4°C, kept on ice, and stimulated as previously described (34). Briefly, thymocytes from NOD, B6, and NOR mice (4 3 107/ml) were suspended in DMEM containing 1 mM Na3VO4 and 1 mg/ml of biotinylated anti-TCR and anti-CD4 mAbs at 4°C. After 15 min, cells were pelleted, resuspended in 1 ml of 5 mg/ml streptavidin that was prewarmed to 37°C, and incubated for the time indicated. Cells were then lysed on ice for 30 min to 1 h with 1% Brij 97 lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 2 mM Na3VO4, 10 mM NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM PMSF and then centrifuged for 10 min at 14,000 rpm at 4°C. Equal amounts of proteins (200 mg/sample) were used for immunoprecipitations.

Cell surface biotinylation Cell surface biotinylation were performed as reported (35), with minor modifications. Briefly, cells were washed three times in PBS and resuspended (107 cells/ml) in PBS containing 0.1 mM CaCl2 and 1 mM MgCl2. Sulfo-N-hydroxysuccinimide-biotin (200 mg/ml; Pierce Chemical Co., Rockford, IL) dissolved in DMSO was added to the cell suspension to a final concentration of 0.5 mg/ml, and the cells were incubated for 1 h on ice. Biotinylated cells were washed three times in RPMI 1640 and lysed in 1% Brij 97 lysis buffer. The sulfonyl group of this biotin derivative confers a net negative charge on the molecule and prevents it from crossing the plasma membrane. This confines the biotinylation reaction to the exoplasmic face of the lipid bilayer and enables the cell surface biotinylation of lysine residues on membrane-associated proteins.

Immunoprecipitation, affinity precipitation, and immunoblotting Postnuclear supernatants of Brij 97 lysates from 1 to 2 3 107 cells were immunoprecipitated (2–16 h) with specific Abs or control isotype-matched preimmune Ig preadsorbed with 30 ml of protein A-Sepharose CL-4B (Pharmacia Biotech, Baie d’Urfe, Canada), protein A/G plus agarose, or protein G plus agarose (Santa Cruz Biotechnology). After incubation, the beads were washed three times in lysis buffer. For affinity precipitation, lysates prepared as described above were incubated with 20 ml of GST, GST-Lck SH2, or SH2/3 domain fusion proteins coupled with agarose beads for 4 h. Bound proteins were solubilized in 23 Laemmli sample buffer under reducing conditions and then transferred onto a polyvinylidene difluoride or nitrocellulose membrane. Immunoblotting was performed after first blocking the membranes with 5% nonfat dry milk in TBS-T (10 mM Tris (pH 7.6), 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature. The membrane was immunoblotted using relevant Abs and was visualized by horseradish peroxidase-conjugated secondary Abs and a chemiluminescence substrate for protein detection (Boehringer Mannheim Canada). Biotinylated proteins were detected using ExAvidin-POD (Sigma) and chemiluminescence. For sequential immunoblotting of a single membrane, the membrane was stripped of proteins for 30 min at 50°C in 62.5 mM Tris (pH 6.7), 2% SDS, and 0.1 M 2-ME and was then immunoblotted. The relative amounts of the proteins detected were quantitated by densitometry using a Molecular Imager (Bio-Rad, Hercules, CA).

Subcellular fractionation Thymocytes (108) were resuspended and lysed by brief sonication in icecold 10 mM Tris (pH 7.4), 10 mM KCl, 1.5 mM MgCl2, and 2 mM EGTA hypotonic buffer containing the protease and phosphatase inhibitors as described above (buffer A). Lysates were adjusted to 150 mM NaCl and centrifuged to remove nuclei and debris, and particulate membrane-containing (P100) and soluble cytoplasm-containing (S100) fractions were separated by differential centrifugation at 100,000 3 g for 30 min. Membrane fractions were washed with ice-cold buffer A and solubilized by sonication in buffer A supplemented with 1% Triton X-100.

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IMPAIRED RECRUITMENT OF CD4-Lck TO THE TCR COMPLEX

FIGURE 1. Different amounts of TCR- and CD4-associated Lck are present in TCR/CD4-stimulated NOD and control thymocytes. Thymocytes (2 3 107) from NOD and B6 mice were stimulated with anti-TCR and anti-CD4 mAbs for 1 min (1) or were left unstimulated (2). After lysis with 1% Brij 97 lysis buffer, cell lysates were precleared and then immunoprecipitated with anti-CD4 (A), anti-TCRb (B), or anti-CD8 (C, upper panel). The supernatants of anti-CD8 immunoprecipitates (C, upper panel) were subsequently immunoprecipitated with anti-CD4 (C, lower panel). Lck associated with CD4, TCR, and CD8 was separated by 10% SDS-PAGE and quantitated by anti-Lck immunoblotting. The membranes in the upper panels of A and C were reprobed with anti-TCRb, and the membrane in the upper panel of B was reprobed with anti-CD4. These immunoblots are shown in the lower panels of A, B, and C. The numbers above each lane represent the OD of the Lck band in arbitrary densitometric units determined by a phosphorimager. The results shown were reproducible in three separate experiments.

Results CD4-associated Lck is sequestered from the TCR complex in activated NOD thymocytes Cross-linking of CD42associated Lck to the TCR and accompanying structural changes in the TCR-CD4-Lck complex are required for efficient TCR signaling in T cells (36). Recruitment of CD4-associated Lck to ligand-engaged TCR determines the agonist and partial agonist properties of peptide-MHC ligands (37). Since the relative amounts of CD4- and TCR-associated Lck regulate the extent of T cell activation, we analyzed whether these respective amounts differ in NOD and control thymocytes before and after TCR/CD4 stimulation. B6 was chosen as a control strain, as B6 thymocytes yield a full proliferative response upon TCR stimulation in vitro (38). The levels of surface expression of CD4 and CD8, respectively, are equivalent on NOD and B6 CD41CD81 double positive and CD41 and CD81 single positive thymocytes, and the total number of thymocytes and the percent distribution of double positive and single positive thymocytes are very similar in NOD and B6 mice (39). In pilot experiments, we did not observe any significant difference in the amounts of total cellular Lck in NOD and B6 thymocytes. To detect quantitative differences in the amounts of CD4-associated Lck, thymocytes were maintained at 4°C, as CD4-Lck association is rapidly induced and re-established in suspension cultures at 37°C (32) (our unpublished observations). Before stimulation, Lck associated with the TCR complex at 4°C, as previously reported (36), and about fourfold more CD4-associated Lck was present in NOD than in B6 thymocytes before and after

stimulation (Fig. 1A, upper panel). However, very low amounts of TCRb were detected in anti-CD4 immunoprecipitates of unstimulated thymocytes (Fig. 1A, lower panel). Interestingly, about twofold less TCRb was observed in anti-CD4 immunoprecipitates of stimulated NOD thymocytes than in B6 thymocytes (Fig. 1A, lower panel). In contrast, an estimated two- to threefold increase in the amount of TCR-associated Lck was observed in stimulated B6 thymocytes compared with NOD thymocytes (Fig. 1B, upper panel). A similar two- to threefold increase in the amount of CD4 was found in anti-TCRb immunoprecipitates of stimulated B6 thymocytes than in those of NOD thymocytes (Fig. 1B, lower panel). Despite the small differences seen in these fold increases in CD4and TCR-associated Lck, the results obtained were highly reproducible in several separate experiments. These observations raise the possibility that TCR/CD4 co-cross-linking induces a diminished recruitment of CD4-associated Lck to the TCR complex in NOD thymocytes. Since double positive thymocytes comprise about 85% of the T cells in a thymus, we investigated whether decreased recruitment of CD4-associated Lck to the TCR complex in stimulated NOD thymocytes is mediated by a concomitant increase in CD8-associated Lck. Relative amounts of CD8-associated Lck in NOD and control thymocytes before and after TCR/CD4 cross-linking were quantified by immunoprecipitation with anti-CD8 and immunoblotting with anti-Lck. Although CD8-associated Lck was detected in NOD and B6 thymocytes after TCR/CD4 cross-linking, it was undetectable in quiescent thymocytes (Fig. 1C, upper panel). A slightly greater amount of CD8-associated Lck was observed in B6

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FIGURE 2. CD4-associated Lck is sequestered from the TCR/CD3 complex in activated NOD thymocytes. A, Experimental protocol used to examine the sequestration of CD4-associated Lck from the TCR/CD3 complex in activated NOD thymocytes. The protocol illustrates the method used for the isolation of TCR-associated Lck (immunoprecipitated with anti-TCRb) and the remaining CD4-associated Lck in the supernatant (immunoprecipitated with anti-CD4) after depletion of TCR-associated Lck. B, Differential association of Lck with TCR and CD4 in thymocytes from 8-wk-old NOD, B6, and NOR mice. Thymocytes from NOD, B6, and NOR mice were stimulated with anti-TCR and anti-CD4 mAbs for 0, 1, and 10 min and were then lysed in 1% Brij 97 lysis buffer. Cell lysates were precleared and immunoprecipitated with an anti-TCRb mAb. Anti-TCRb immunoprecipitates were subjected to 10% SDS-PAGE, and the amount of TCR-associated Lck was estimated by anti-Lck immunoblotting (upper panel). The efficiency of preclearing was confirmed by the absence of TCRb in the second anti-TCRb immunoprecipitate (middle panel). Supernatants of the immunoprecipitates shown in the middle panel of B were subsequently immunoprecipitated with an anti-CD4 mAb. The amount of CD4-associated Lck was detected as described for the upper panel of B. C, Association of Lck with TCR and CD4 in thymocytes from 3-wk-old NOD and B6 mice. Experiments similar to those described in B were performed. Relative band intensities were determined by densitometry. The numbers above each lane represent the OD of the Lck band in arbitrary densitometric units determined by a phosphorimager. The results shown were reproducible in three separate experiments.

thymocytes. This CD8-associated Lck does seem to be associated with the TCR complex, as reprobing the same membrane with an anti-TCRb Ab did not show any detectable band corresponding to TCRb (Fig. 1C, middle panel). Dissociation of Lck from CD4 did not give rise to the induced association of Lck with CD8, since the amounts of CD4-associated Lck observed in stimulated B6 and NOD thymocytes (Fig. 1C, lower panel) were very similar to those shown in Fig. 1A. This suggests that decreased recruitment of CD4-associated Lck to the TCR complex in stimulated NOD thymocytes is not mediated by an increase in the amount of CD8-associated Lck. Since recent evidence suggests that CD4 cross-linking may inhibit T cell activation by the sequestration of CD4-associated Lck (26, 27), we determined whether this sequestration occurs after TCR stimulation of NOD thymocytes. The amounts of TCR- and CD4-associated Lck in NOD, B6, and NOR thymocytes were assayed after depletion of TCR-associated Lck (Fig. 2A). NOR was included as another control strain, since although NOR mice are MHC matched and congenic with NOD mice at several chromosomal regions, NOR mice are diabetes resistant (40). Like NOD mice, NOR mice develop insulitis (40), and their T cells are hyporesponsive to TCR stimulation of proliferation (our unpublished observations).

At 1 to 10 min after TCR/CD4 co-cross-linking, significantly more Lck was recruited to the TCR complex in B6 thymocytes than in either NOD or NOR thymocytes (Fig. 2B, upper panel). No detectable TCRb was observed in a second immunoprecipitation with anti-TCRb (Fig. 2B, middle panel), suggesting that most of the TCR complex was effectively removed in the first immunoprecipitation. In contrast, more CD4-associated Lck was present in NOD thymocytes than in control NOR and B6 thymocytes both in the quiescent state (0 min) and after 1 and 10 min of TCR/CD4 co-cross-linking (Fig. 2B, lower panel). The low amounts of TCRassociated Lck observed in stimulated NOD thymocytes correlate to the high levels of CD4-associated Lck. These results show that CD4-associated Lck is sequestered from the TCR complex in activated NOD thymocytes but not in control B6 activated thymocytes, suggesting that NOD thymocyte proliferative hyporesponsiveness is mediated by the impaired recruitment of CD4associated Lck to the TCR complex. Interestingly, after 1 min of TCR/CD4 co-cross-linking, dissociation of Lck from CD4 is evident in NOR thymocytes, but the amount of TCR-associated Lck does not increase reciprocally. Presently, we do not know which molecule(s) this dissociated Lck becomes associated with in activated NOR thymocytes. Together, these findings indicate that the proliferative hyporesponsiveness of

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FIGURE 3. An excess of free membrane-associated CD3ge and TCRz chains is present in NOD thymocytes relative to that in B6 thymocytes. A, Thymocytes from NOD and B6 mice were surface labeled with sulfo-N-hydroxysuccinimide-biotin (NHS) for 1 h on ice and were then lysed in 1% Brij 97 lysis buffer. Cell lysates were sequentially immunoprecipitated twice with anti-TCRb and then with anti-CD3e, resolved by 15% SDS-PAGE, and transferred to a nitrocellulose membrane. The biotinylated proteins were detected using ExAvidin-peroxidase and chemiluminescence (A). The numbers above each lane represent the optical density of the TCRab, CD3ge, and TCRz bands in arbitrary densitometric units determined by a phosphorimager. B, CD3ge chains were confirmed by subsequent reprobing of the membrane with anti-CD3g, anti-CD3d, anti-CD3e, and anti-TCRz Abs. The results shown were reproducible in two separate experiments. C, Thymocytes (108/sample) from NOD and B6 mice were stimulated with anti-TCR and anti-CD4 mAbs and were then lysed in hypotonic lysis buffer. Membrane fractions were precleared twice with an anti-TCRb mAb, and the efficiency of preclearing was monitored by the absence of TCRb in the second anti-TCRb immunoprecipitate (lower panel). D, The supernatants were further immunoprecipitated with an anti-CD3e mAb and blotted with an anti-phosphotyrosine mAb (upper panel). The positions of the CD3e and TCRz chains were confirmed by immunoblotting with anti-CD3e and anti-TCRz Abs (middle and lower panels). E, The membrane was stripped and reprobed with an anti-Lck mAb.

NOD and NOR thymocytes appear to be mediated by similar, yet different, TCR-dependent signaling events. Previously, we reported that T cell hyporesponsiveness in NOD mice occurs only after the first 5 to 6 wk of life (28, 29). To further determine whether NOD T cell hyporesponsiveness is indeed mediated by the impaired recruitment of CD4-associated Lck to the TCR complex, we analyzed whether the association of CD4-Lck with the TCR in young (,5– 6 wk-old) NOD mice is more like that observed in B6 thymocytes. Thymocytes from 3-wk-old NOD and age-matched B6 female mice were stimulated, lysed, immunoprecipitated, and immunoblotted as described above in Figure 2B. The amounts of both TCR-associated and remaining CD4-associated Lck in unstimulated NOD and B6 thymocytes were similar, and after TCR/CD4 co-cross-linking most of the CD4-associated Lck was recruited to the TCR complex in both NOD and B6 thymocytes (Fig. 2C, upper and lower panels). These data further support the idea that impaired recruitment of CD4-associated Lck to the TCR complex plays an important role in the induction of T cell hyporesponsiveness. Free TCRz and CD3ge chains bind more to Lck in NOD than in B6 thymocytes The mechanism of sequestration of CD4-associated Lck from the TCR complex in NOD thymocytes and its effect on downstream signaling events were analyzed. We tested whether the binding of

CD4-associated Lck to an excess of free CD3 and TCRz chains may preclude its juxtaposition to the TCR complex in stimulated NOD thymocytes. This reasoning is based on the report that an excess of TCRz chains is synthesized in both thymocytes and splenic T cells (41), and that TCRz as well as CD3g, -d, and -e chains can associate with the plasma membrane independently of TCRab (42). To determine whether an excess of TCRz and CD3 chains accumulates on the plasma membrane of NOD thymocytes, these cells were surface biotinylated, lysed, and then serially immunoprecipitated with anti-TCRb and anti-CD3e to recover and quantitate the distribution of all plasma membrane-bound TCRz and CD3 chains. The levels of expression of TCRab and TCRz were similar in NOD and B6 thymocytes, as the ratio of TCRab: CD3ge:TCRz was 4.0:2.7:1 in NOD thymocytes and 3.3:2.2:1 in B6 thymocytes in the first anti-TCRb immunoprecipitate (Fig. 3A). The expression of CD3g, CD3e, and TCRz, but not that of CD3d, was confirmed by reprobing the membrane with anti-CD3g, anti-CD3d, and anti-CD3e Abs (Fig. 3B). Interestingly, although TCRab was virtually absent after a second immunoprecipitation with anti-CD3e, CD3g, CD3e, and TCRz chains were still detectable, particularly in NOD thymocytes. Similar results were observed in splenic T cells (our unpublished observations). Thus, CD3g, CD3e, and TCRz chains can associate with the plasma membrane independently of TCRab, as previously reported (35,

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FIGURE 4. The binding of free CD3ge and TCRz chains to CD4-associated Lck is greater in NOD than in B6 thymocytes. A, Thymocytes were surface biotinylated; incubated with anti-TCR and anti-CD4 mAbs on ice for 15 min; cross-linked by rabbit anti-hamster Ig for 0, 1, 10, and 30 min; and then lysed in 1% Brij 97 lysis buffer. After preclearing TCR-associated TCRz chains by immunoprecipitation twice with an anti-TCRb mAb, supernatants were further immunoprecipitated with an anti-CD4 mAb. Biotinylated proteins were detected by ExAvidin-POD and chemiluminescence. The numbers above each lane represent the OD of the CD4, CD3ge, and TCRz bands in arbitrary densitometric units determined by a phosphorimager (upper panel). The same membrane was stripped and reprobed with anti-CD3e and anti-TCRz Abs (middle and lower panels). B, NOD thymocytes were surface biotinylated, incubated with anti-TCR and anti-CD4 mAbs on ice for 15 min, cross-linked with rabbit anti-hamster Ig at 37°C for 1 min, and then lysed as described above. The cell lysates were precleared with protein A/G-agarose, and the TCR/CD3 complex was depleted by further preclearing with anti-TCRb. The supernatants were incubated with GST Lck-SH2, GST Lck-SH2/3, or GST alone to remove membrane-associated CD3 and TCRz chains and were further immunoprecipitated with an anti-CD4 mAb. CD4-associated biotinylated proteins were detected as described in A. Results shown were reproducible in two separate experiments.

42). These free, membrane-associated, CD3g, CD3e, and TCRz chains are more abundant in NOD than in B6 thymocytes. Next, we determined whether these free CD3ge and TCRz chains were tyrosine phosphorylated after TCR/CD4 co-crosslinking or if they were constitutively phosphorylated, since tyrosine phosphorylation of TCRz and CD3ge chains is an important step leading to downstream signaling events. Thymocytes from NOD and B6 mice were stimulated as described in Figure 2 and lysed in hypotonic lysis buffer. Membrane fractions were precleared twice with anti-TCRb mAb, and anti-TCRb immunoblotting of the two anti-TCRb immunoprecipitates showed equal amounts of TCRb in the first precipitates (Fig. 3C, upper panel) and no detectable TCRb remaining in the second precipitates (Fig. 3C, middle panel). After further precipitation of the precleared lysates with anti-CD3e and immunoblotting with antiphosphotyrosine, no detectable tyrosine phosphorylation of CD3e and TCRz was found in unstimulated NOD and B6 thymocytes (Fig. 3D, upper panel). However, TCR/CD4 stimulation induced an increase in tyrosine phosphorylation of CD3e and TCRz in both NOD and B6 thymocytes, with the amount of phosphorylation being about threefold greater in NOD thymocytes than in B6 thymocytes. Reprobing the filters with anti-CD3e and anti-TCRz Abs showed that about three- to fourfold more CD3e and TCRz were present in NOD than in B6 thymocytes before and after stimulation (Fig. 3D, middle and lower panels). To identify which PTK phosphorylates these free CD3ge and TCRz chains, TCR-precleared anti-CD3e immunoprecipitates were immunoblotted with anti-Lck and anti-Fyn. After TCR/CD4 cross-linking, about threefold more Lck was present in these precipitates of NOD thymocytes than in those of B6 thymocytes (Fig.

3E). No Fyn was detected in these precipitates (data not shown). These results indicate that TCR/CD4 co-cross-linking induces the tyrosine phosphorylation of free TCRz and CD3ge, most likely by Lck. Since TCRz is an endogenous substrate for Lck (9), we investigated whether excess TCRz chains bind to CD4-associated Lck on the plasma membrane. NOD and B6 thymocytes were surface biotinylated and then stimulated with anti-TCR and anti-CD4 mAbs for 0, 1, 10, and 30 min. After preclearing cell lysates of the TCR by immunoprecipitation with anti-TCRb, the lysates were immunoprecipitated with anti-CD4 and visualized with ExAvidinPOD and chemiluminescence. Binding of TCRz and CD3ge to CD4-Lck was evident 1 min after stimulation in NOD but not in B6 thymocytes, and this binding was not apparent after longer periods of stimulation (Fig. 4A, upper panel). The presence of CD3e and TCRz in anti-CD4 immunoprecipitates of NOD thymocytes was confirmed by immunoblotting with anti-TCRz (Fig. 4A, middle and lower panels). To further test whether free, plasma membrane-associated TCRz, CD3g and CD3e chains bind to CD4-associated Lck, a competition experiment was performed in which NOD thymocytes were surface biotinylated, stimulated with anti-TCR plus anti-CD4 for 1 min, and lysed as described above. Figure 4B illustrates that incubation of anti-TCRb precleared NOD thymocyte lysates with Lck-SH2 and Lck-SH2/SH3 fusion proteins depleted most ($90%) of the membrane-associated TCRz and CD3ge chains. The efficiency of preclearing was confirmed by the absence of detectable TCRb in the second anti-TCRb precipitation (data not shown). Subsequent immunoprecipitation of these lysates with antiCD4 yielded considerably less membrane-associated TCRz and

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FIGURE 5. Interaction between Lck and free TCRz chains is mediated by the Lck SH2 domain. A, Thymocytes from NOD and B6 mice were stimulated with anti-TCR plus anti-CD4 mAbs for 10 min and then lysed in 1% Brij 97 lysis buffer. Cell lysates were immunoprecipitated twice with anti-TCRb to remove TCR/CD3-associated TCRz chains. The efficiency of preclearing was monitored by anti-TCRb immunoblotting based on the absence of TCRb in the second anti-TCRb precipitate (middle panel). B, The supernatants were further incubated with GST, GST Lck-SH2, and GST Lck-SH2/3 domain fusion proteins coupled to agarose beads, separated by 15% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-TCRz polyclonal Abs. NOD thymocytes were stimulated, lysed, and precleared as described in A. However, the sample in lane 2 was not precleared with anti-TCRb and served as a positive control. The sample in lane 5 was also not precleared with anti-TCRb, but rather was immunoprecipitated with normal rabbit serum (NRS) as a negative control. C, After incubation with GST, GST Lck-SH2, or GST Lck-SH2/3 domain fusion proteins coupled to agarose beads, the supernatants were precleared with anti-TCRb, further immunoprecipitated with anti-Lck polyclonal Abs, resolved on 15% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-TCRz polyclonal Abs. The second anti-TCRb immunoprecipitates were blotted with anti-TCRb to monitor the efficiency of preclearing (lower panel). Results shown were reproducible in three separate experiments.

CD3ge chains than that observed in a control sample preincubated with the GST protein alone. Thus, free plasma membrane-associated TCRz and CD3ge chains can bind to CD4-associated Lck and possibly be phosphorylated by CD4-associated Lck. Interaction between Lck and excess TCRz in activated NOD thymocytes is mediated by the SH2 domain of Lck Signaling protein interactions are often mediated through the binding of SH2 domains to phosphotyrosine residues in many PTKs, protein tyrosine phosphatases, and adaptor proteins (43– 45). However, the binding of tyrosine-phosphorylated TCRz to Lck may require both the SH2 and SH3 domains of Lck (44). Our results in Figure 4B implicated a role for the SH2 domain of Lck in the association of CD4 with free membrane-associated TCRz, CD3g, and CD3e chains in NOD thymocytes. To further investigate this role, we examined whether excess TCRz chains can bind to only the SH2 domain or to both the SH2 and SH3 domains of Lck. After TCR and CD4 costimulation of NOD thymocytes, lysates were precleared of TCR-associated TCRz by immunoprecipitation with anti-TCRb. Equal amounts of TCRb were seen in the first round of anti-TCRb immunoprecipitates (Fig. 5A, upper panel), but no detectable TCRb was found in the second round of immunoprecipitates (Fig. 5A, lower panel). Precleared cell lysates were then incubated with either GST or the GST Lck SH2 and SH2/SH3 fusion proteins. The latter fusion proteins bound to significantly greater amounts of TCRz in NOD than in B6 thymocytes (Fig. 5B). A competition experiment in which anti-TCRb-precleared lysates of stimulated NOD thymocytes were incubated with these

fusion proteins and then immunoprecipitated with anti-Lck revealed that both these fusion proteins completely inhibited the binding of Lck to TCRz (Fig. 5C, upper panel). Efficient preclearing of TCRb from the lysates was observed (Fig. 5C, lower panel). These data are consistent with the idea that the association between Lck and excess free TCRz, and CD3g and CD3e chains may occur through the SH2 domain of Lck.

Discussion Several observations suggest that CD4 associates with the TCR complex during T cell activation, and that this association is not only dependent on Lck but also is critical for the CD4-mediated enhancement of T cell responsiveness (46). In this study we examined the activation of CD4- and TCR-associated Lck after the engagement of these surface receptors in thymocytes from autoimmmune diabetes-prone NOD mice and diabetes-resistant control mice. We provide evidence that NOD T cell proliferative hyporesponsiveness may be mediated by the sequestration of Lck by CD4 and the impaired recruitment of CD4-associated Lck to the TCR complex. Our findings demonstrate that in TCR-activated NOD thymocytes, a significant proportion of membrane-bound CD4-associated Lck molecules binds to the TCRz and CD3ge chains present in relative excess in these cells. Based on these findings, we propose a model that illustrates a mechanism of how this CD4-Lck-TCRz/ CD3ge interaction may result in only a partial downstream signal for NOD thymocyte activation (Fig. 6). It is important to mention

The Journal of Immunology that this model represents one of several possible models of differential signaling by the TCR, as additional kinetic and conformational models of partial TCR activation and T cell stimulation were recently described (reviewed in Ref. 46). In our model, upon TCR/CD4 co-cross-linking, most CD4-associated Lck molecules normally move close to the TCR complex in activated T cells. CD4-associated Lck phosphorylates CD3ge subunits and TCRz chains, and the phospho-TCRz-mediated recruitment of another PTK, ZAP-70, to the TCR/CD3 complex then elicits a full downstream signal for T cell activation. Possibly due to the higher expression of free membrane-associated TCRz and CD3ge chains, activated CD4-associated Lck binds preferentially to these chains on the surface of NOD thymocytes. Thus, sequestration of Lck by CD4 in activated NOD thymocytes correlates with the increased binding of CD4-associated Lck to apparently free, membrane-associated TCRz and CD3ge chains. This may diminish the recruitment of Lck to the TCR complex in NOD thymocytes, and this block in the association of CD4-Lck with the TCR complex may elicit a partial downstream signal for T cell activation. Further support for the diminished recruitment of Lck to the TCR complex is provided by our finding (Fig. 1C) that association of Lck with CD8 induced by TCR/CD4 co-cross-linking occurs independently of the TCR complex. Although CD4 enhances Ag-mediated activation of T cells by binding to MHC class II, CD4 cross-linking can also block subsequent Ag-induced or anti-TCR-induced T cell activation. This indicates that CD4 may also transduce a negative signal to the TCR, possibly as a result of the sequestration of Lck by CD4 (24 –27). Furthermore, CD4 ligands can prevent the association among phospholipase C-g1, p120GAP, and other signaling molecules, and this may result in the formation of defective multifunctional complexes and the inhibition of T cell activation (47). Notwithstanding, direct biochemical evidence for CD4 sequestration of Lck has not yet been reported. We reasoned that if CD4-associated Lck does not associate appropriately with the TCR complex in activated NOD thymocytes, this might inhibit TCR-dependent downstream signaling. Our data show that following co-cross-linking of TCR and CD4, the relative amount of TCR-associated Lck present in NOD thymocytes is lower than that found in control

FIGURE 6. Model of sequestration of CD4associated Lck from the TCR/CD3 complex in NOD thymocytes. Following TCR-induced activation of NOD thymocytes, some CD4-associated Lck molecules are sequestered by binding to an excess of free TCRz and CD3ge chains on the plasma membrane. This may prevent or diminish the recruitment of Lck to the TCR, result in the impaired coupling of the TCR to downstream signaling events, and lead to T cell proliferative hyporesponsiveness.

1155 strain thymocytes. In contrast, the amount of CD4-associated Lck was significantly increased in activated NOD thymocytes. It is unlikely that the dissociation of Lck from CD4 observed in activated control B6 and NOR thymocytes occurred following cell lysis, as no evidence for dissociation of Lck from CD4 was detected in similarly lysed, activated NOD thymocytes. Moreover, very similar levels of association of Lck with CD4 were observed on the plasma membrane of NOD and B6 thymocytes after incubation for 45 min at 37°C (our unpublished observations). These findings suggest that in TCR-activated NOD thymocytes much of the CD4-associated Lck remains physically sequestered from the TCR complex. Interestingly, this sequestration of CD4-associated Lck from the TCR complex does not occur in thymocytes from 3-wk-old NOD mice, suggesting that impaired recruitment of CD4-associated Lck to the TCR complex may play an important role in initiating T cell hyporesponsiveness in NOD mice. Many properties of TCRz chains distinguish them from other components of the TCR complex. All synthesized TCRz chains are efficiently coupled to a partially assembled TCR complex, and TCRz chains determine the rate and fate of assembly and transport to the plasma membrane of a complete TCR complex (48). TCRz chains, but not other components of the surface TCR complex, may be rapidly exchanged in this complex with newly synthesized TCRz chains (34). Moreover, free CD3 chains, which are not assembled to the TCR complex, may also associate directly with the plasma membrane (42). Using the approach of cell surface biotinylation followed by serial immunoprecipitation, we found that both free TCRz and CD3ge chains associate with the plasma membrane independently of TCRab in activated NOD thymocytes. Notably, we detected a relatively greater amount of free membraneassociated TCRz and CD3ge chains in NOD thymocytes than in B6 thymocytes. These free membrane-associated TCRz as well as CD3g and CD3e chains represent a potential target of interaction with Lck. Indeed, we observed higher levels of tyrosine phosphorylation of these free membrane-associated CD3ge and TCRz chains after stimulation in NOD thymocytes than in B6 thymocytes. Note that increased levels of tyrosine phosphorylation of free CD3ge and TCRz chains in NOD thymocytes correlate to the high amounts of Lck observed in these TCR-precleared anti-CD3e

1156


immunoprecipitates. Therefore, one mechanism of sequestration of CD4-associated Lck from the TCR complex in NOD thymocytes may involve the enhanced binding of free TCRz and CD3ge chains to the SH2 domain of Lck. This binding may significantly reduce the amount of CD4-associated Lck available to be recruited to and associate, via its SH2 domain, with the TCR complex. TCRz, ZAP-70, and Syk can associate with Lck, and this association may be blocked by a peptide corresponding to the Lck SH2 domain binding site (49), suggesting that the Lck SH2 domain binds directly to tyrosine-phosphorylated ZAP-70 and that the association of TCRz with Lck may be indirectly coupled via ZAP-70 (49). However, Lck can bind directly to phospho-TCRz, and this interaction requires both the SH2 and SH3 domains of Lck (44). Our studies show that CD4-associated Lck and free TCRz and CD3ge chains physically associate with each other on the plasma membrane after TCR/CD4 co-cross-linking in NOD thymocytes. They also demonstrate that the SH2 domain of Lck may play an important role in regulating the interaction between Lck and excess free TCRz, CD3g, and CD3e chains. Thus, sequestration of Lck by CD4 in activated NOD thymocytes would appear to correlate with the increased binding of CD4-associated Lck via its SH2 domain to apparently free, membrane-associated TCRz and CD3ge chains. This proposed role for the Lck SH2 domain in association with TCRz is consistent with the observation that the Lck SH2 GST fusion protein directly interacts with TCRz and that this interaction is enhanced upon TCR stimulation (45). We failed to observe phospho-ZAP-70 associated with Lck after TCR/CD4 cross-linking, suggesting that the association between Lck and ZAP-70 may be either indirect or cell type dependent. Ligation of CD4 by various ligands, such as HIV and gp120, generally induces the dissociation of Lck from CD4 and leads to TCR desensitization (27, 50, 51). Presumably, our ability to detect and quantify the sequestration of Lck by CD4 on the plasma membrane of thymocytes was facilitated by two experimental conditions. First, we conducted related experiments at 4°C rather than 37°C, as after ligand-induced dissociation from CD4, Lck reassociates with CD4 very rapidly at 37°C (33). Second, we monitored changes in the amounts of CD4- and TCR-associated Lck relatively early (1–10 min) after TCR/CD4 co-cross-linking of thymocytes. By comparison, if CD4 is engaged by gp120/anti-gp120 cross-linking for 1 to 4 h at 37°C, Lck is dissociated from CD4 and is then translocated to and sequestered in the cytoskeleton in association with actin (27). This cytoskeletal sequestration of Lck results in TCR signaling defects. Additional studies are underway to investigate whether Lck that is initially sequestered by CD4 in the plasma membrane is then sequestered in the cytoskeleton of NOD thymocytes. In conclusion, our data provide the first direct biochemical evidence that Lck is sequestered by CD4 from the TCR in T cells after stimulation through the TCR, and that this correlates with the binding of CD4-associated Lck to free plasma membrane-associated TCRz and CD3ge chains. It is of interest that Lck sequestration, as reflected by the relative increases in the amount and the activity of CD4-associated Lck, was detected in activated thymocytes from autoimmune diabetes-susceptible NOD mice. It remains to be determined whether, and if so how, these increases in CD4-associated Lck regulate the traits of T cell proliferative hyporesponsiveness and susceptibility to diabetes in NOD mice. Notwithstanding, we found that the elevated amount of CD4-associated Lck is accompanied by a significantly decreased amount of TCR-associated Lck in NOD thymocytes. Conceivably, diminished recruitment of CD4-associated Lck to the TCR complex results in the CD4-mediated sequestration of Lck from the TCR and the consequent impaired coupling of CD4-Lck to the TCR com-

plex. Taken together, our data suggest that the apparent binding of CD4-associated Lck via its SH2 domain to free TCRz and CD3ge chains on the plasma membrane may mediate the reduced coupling of CD4-Lck to the TCR complex in NOD thymocytes.

Acknowledgments We thank Drs. A. Veillette, A. Singer, R. Alexander, and L. Samelson for their generous donation of reagents; Drs. S. Kaga, B. Gill, J. Madrenas, and A. Veillette for their helpful suggestions and criticisms; all members of our laboratory for their valuable advice and encouragement; Ms. A. Leaist for her expert and cheerful assistance with the preparation of this manuscript; and Ms. C. Richardson and the Robarts Animal Facility staff for the breeding and maintenance of the mice used in these studies.

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