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The Journal of Immunology, 2002, 169: 230-238.
Copyright © 2002 by The American Association of Immunologists

A Therapeutic CD4 Monoclonal Antibody Inhibits TCR-{zeta} Chain Phosphorylation, {zeta}-Associated Protein of 70-kDa Tyr319 Phosphorylation, and TCR Internalization in Primary Human T Cells1

Susanne Harding*, Peter Lipp{dagger} and Denis R. Alexander2,*

Laboratories of * Lymphocyte Signaling and Development and {dagger} Molecular Signaling, The Babraham Institute, Cambridge, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The molecular mechanisms mediating the inhibitory effects of a humanized CD4 mAb YHB.46 on primary human CD4+ T cells were investigated. Preincubation of T cells with soluble YHB.46 caused a general inhibition of TCR-stimulated protein tyrosine phosphorylation events, including a reduction in phosphorylation of p95vav, linker for activation of T cells, and Src homology 2 domain-containing leukocyte protein of 76-kDa signaling molecules. A marked reduction in activation of the Ras/mitogen-activated protein kinase pathway was also observed. Examination of the earliest initiation events of TCR signal transduction showed that YHB.46 inhibited TCR-{zeta} chain phosphorylation together with recruitment and tyrosine phosphorylation of the {zeta}-associated protein of 70-kDa tyrosine kinase, particularly at Tyr319, as well as reduced recruitment of p56lck to the TCR-{zeta} and {zeta}-associated protein of 70-kDa complex. These inhibitory events were associated with inhibition of TCR endocytosis. Our results show that the YHB.46 mAb is a powerful inhibitor of the early initiating events of TCR signal transduction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4 is a 55-kDa integral membrane glycoprotein that binds to nonpolymorphic regions on MHC class II molecules (1, 2, 3) that simultaneously engage the TCR (3, 4), resulting in the coaggregation of CD4 with the TCR (5, 6). The CD4 cytoplasmic tail binds noncovalently to the p56lck tyrosine kinase (7, 8) that is thought to facilitate the physical coassociation of the TCR and its CD4 coreceptor (9). TCR signaling cascades are initiated upon CD4/p56lck-TCR aggregation by phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM)3 motifs located in the TCR-{zeta} and CD3-{epsilon} chains (10, 11). Consistent with this model, p56lck is enriched within the central domain of the supramolecular activation clusters that are generated following MHC-peptide binding to the TCR (12). Furthermore, coligation of CD4 with the TCR enhances T cell responses (13, 14, 15, 16).

Phosphorylation of TCR-{zeta} and CD3 polypeptide ITAM motifs results in the recruitment of the {zeta}-associated protein of 70-kDa (ZAP-70) tyrosine kinase and its subsequent phosphorylation and activation by kinases such as p56lck (17, 18, 19). These events are regulated by the CD45 phosphotyrosine phosphatase that has both positive and negative roles in regulating p56lck (20, 21, 22). p56lck is thought to bind Tyr319 within the linker region of ZAP-70 and as a consequence greatly increase the actions of this kinase (23, 24). Several important adaptor proteins lie downstream of ZAP-70 activation (25, 26), such as linker for activation of T cells (LAT) (27), Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) (28, 29), and p95vav (Vav) (30, 31), that in turn couple the TCR to the Ras/mitogen-activated protein kinase (MAPK) and calcium signaling pathways that regulate the transcription factors responsible for IL-2 gene induction (32). Stimulated TCRs that induce these pathways are internalized by a process of "serial engagement," thought to be important for the progression of T cell activation events (33, 34).

Engagement of CD4 by mAbs without concomitant cross-linking to the TCR causes inhibition of TCR signal transduction (35, 36) by a process that can be independent of the presence of APC-expressing MHC class II (37). CD4 mAbs directed against a CD4 ectodomain fail to block MHC class II tetramer binding to Ag-specific T cells, yet inhibit activation, further suggesting that CD4 engagement results in the delivery of negative signals to T cells independently of blocking effects on CD4-class II interactions (38, 39). Negative effects of CD4 mAbs on TCR-stimulated downstream signaling pathways have been described, including inhibition of signal transduction complexes (40), calcium signals (41, 42), extracellular signal-related kinase (Erk)2 and c-Jun N-terminal kinase activation (43, 44), and transcription factors important for IL-2 gene induction, such as NF-AT, NF-{kappa}B, and AP-1 (44, 45). Such findings suggest an inhibitory locus of action of CD4 mAbs very early in the signaling cascades that result in T cell activation. Surprisingly, relatively little information is available about the inhibitory effects of CD4 mAbs on the very earliest events of TCR signal transduction coupling. Murine CD4+ T cell clones exposed to wild-type ligand in the presence of inhibitory CD4 mAb display a pattern of signaling similar to that seen with partial agonists, with reduced TCR-{zeta} phosphorylation and no detectable phosphorylation of ZAP-70 (46). Whether CD4 mAbs have such effects in primary resting human T cells has not been reported. Furthermore, the role of CD4-associated p56lck in the inhibitory process remains controversial. An attractive model for inhibition is that ligation of CD4 by mAb-binding sequesters p56lck away from the TCR, thereby lowering the intensity of TCR signal transduction by decreasing TCR-{zeta} and CD3-{epsilon} phosphorylation (46, 47, 48). However, p56lck-independent inhibitory effects of CD4 mAbs have also been demonstrated by reconstituting T cell clones or hybridomas with CD4 mutants unable to bind p56lck (49, 50), casting doubt on the sequestration hypothesis, at least in preactivated cells.

Recently, we have described the inhibitory effects of a humanized therapeutic nondepleting CD4 mAb YHB.46 (51) on the activation of primary human T cells (52). This CD4 mAb inhibited CD3-stimulated proliferation and the expression of the CD25, CD40 ligand, and CD69 activation markers, with a strikingly reduced production of IL-2, IL-4, and IL-10. The inhibitory effects of YHB.46 were equipotent in the presence or absence of CTLA-4 Ig, suggesting that the inhibitory effects were exerted on TCR-induced rather than CD28-triggered signaling pathways (52). In the present study, we describe the molecular effects of YHB.46 on the earliest initiation events of TCR signal transduction coupling and demonstrate that the CD4 mAb inhibits TCR internalization.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

Abs used were specific for CD11b (OKM1), CD8 (Sigma-Aldrich, Dorset, U.K.), CD3 (UCHT1), CD14, CD16, CD33, and glycophorin (Serotec, Oxford, U.K.), CD19 (BU12; from Prof. P. Beverley, The Jenner Institute, Compton, U.K.), and HLA-DR (BD PharMingen, Oxford, U.K.). The CD3 mAb OKT3 was a gift from Janssen-CILAG (High Wycombe, U.K.). The YHB.46 CD4 mAb (53) was from Dr. G. Hale (Therapeutic Ab Center, Oxford, U.K.). 2G6, a humanized mouse lysozyme isotypic control Ab to YHB.46, was a gift from Dr. R. Field (Cambridge Ab Technology, Royston, U.K.). Immunoprecipitating Abs used were Fb2 phosphotyrosine mAb (from Dr. D. Cantrell, Imperial Cancer Research Fund, London, U.K.), Vav antiserum (from Dr. M. Turner, The Babraham Institute, Cambridge, U.K.), goat anti-{zeta} N39 (CT-11) antiserum (from Prof. C. Terhorst, Boston, MA), ZAP-70 rabbit polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-ZAP-70 rabbit polyclonal Ab (Cell Signaling, Beverly, MA), and rabbit polyclonal LAT (Upstate Biotechnology, Lake Placid, NY). Western blotting Abs were used against phosphotyrosine (4G10; Upstate Biotechnology), Erk1 (Santa Cruz Biotechnology), Erk2 (Transduction Laboratories, Oxford, U.K.), phospho-p44/42 MAPK (Cell Signaling), p56lck (from Dr. L. Samelson, National Institutes of Health, Bethesda, MD), SLP-76 (from Dr. G. Koretzky, University of Pennsylvania Medical School, Philadelphia, PA), Vav (Upstate Biotechnology), ZAP-70 (mAb from GlaxoSmithKline, Stevenage, U.K.), and TCR-{zeta} chain (Zymed Laboratories, San Francisco, CA). HRP-conjugated secondary Abs were from DAKO (Ely, U.K.). Abs used for confocal imaging microscopy were UCHT1-biotin (Serotec), anti-human IgG FITC conjugate (Sigma-Aldrich), and streptavidin-Texas Red from Amersham Pharmacia Biotech (Little Chalfont, U.K.).

Cells

Primary CD4+ T lymphocytes were purified from buffy coats obtained from random blood donors to the National Blood Transfusion Service (Addenbrooke’s Hospital, Cambridge, U.K.). PBMC isolated by Lymphoprep (Nycomed, Oslo, Norway) density gradient centrifugation were depleted of adherent cells by incubation in plastic tissue culture flasks for 45 min at 37°C. Isolation of CD4+ T cells was then undertaken by immunomagnetic negative selection. Cells were resuspended in RPMI 1640 medium (Life Technologies, Paisley, U.K.) supplemented with antibiotics (penicillin and streptomycin), L-glutamine and 5% FCS, and incubated for 30 min at 4°C with saturating concentrations of CD8, CD11b, CD14, CD16, CD19, CD33, HLA-DR, and glycophorin mAbs. The cells were washed twice and incubated with sheep anti-mouse IgG-coupled Dynabeads (Dynal Biotech, Oslo, Norway) for 30 min at 4°C. Monocytes, NK cells, B cells, dendritic cells, erthyrocytes, and CD8+ cells were then removed by incubation of the cell suspension with a Dynal magnetic particle concentrator for 2 min. The supernatant containing nonmagnetized cells was removed and the selection process repeated. The cells were washed in RPMI 1640 medium supplemented with antibiotics and L-glutamine and allowed to rest for 60 min at 37°C. Cell suspensions were routinely checked by flow cytometry and found to be at least 85% pure CD3+CD4+CD8- T cells.

Cell stimulation and immunoprecipitation

CD4+ T cells (5 x 107 cells) were incubated for 10 min at 37°C in the presence of the CD4 mAb (YHB.46) or vehicle (PBS). Cell suspensions were then washed once before stimulation with CD3 mAb (UCHT1) immobilized onto 0.1-µm latex beads (Sigma-Aldrich) for 5 min at 37°C. Activation was stopped by pelleting cells and lysis for 10 min at 4°C in 1% Nonidet P-40, 20 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EGTA, 1 mM sodium orthovanadate, and protease inhibitors (protease inhibitor cocktail tablets; Roche Molecular Biochemicals, Sussex, U.K.). Nuclei and cellular debris were removed by centrifugation for 10 min at 10,000 rpm at 4°C. For analysis of protein tyrosine phosphorylation, samples (equivalent to 106 cells) were added to Laemmli’s buffer and boiled for 5 min. The lysates were precleared by rotating at 4°C for 30–60 min with 40 µl 50% packed protein G-Sepharose beads (Amersham Pharmacia Biotech). Precleared lysates were incubated at 4°C for 60 min or overnight with immunoprecipitating Ab. Immune complexes were recovered by incubation with 40 µl 50% packed protein G-Sepharose beads for 1 h at 4°C. Pellets were washed three times with lysis buffer, resuspended in Laemmli’s buffer, and boiled for 5 min.

Western blotting

Total lysates or immunoprecipitates were separated by SDS-PAGE and electrophoretically transferred onto Immobilon-P. Membranes were blocked for 60 min or overnight in 5% Marvel milk powder in TBST, followed by incubation with specific Abs for 60 min. Membranes were then washed three times in TBST before the addition of HRP-linked secondary Ab. Proteins were visualized by ECL (Amersham Pharmacia Biotech). Reprobing was performed after stripping the membrane by incubation at 55°C in 62.5 mM Tris (pH 6.7), 2% SDS, and 100 mM 2-ME for 30 min with occasional agitation. Quantification of immunoblots was conducted using a phosphorimager (Fuji FLA3000; Raytest, Straubenhardt, Germany).

In vitro kinase assay

Immune complexes were prepared as described above and washed once in kinase buffer (50 mM PIPES (pH 7.4), 10 mM MnCl2, 10 mM MgCl2, 1 mM sodium orthovanadate, and 1 mM AEBSF). The kinase reaction was initiated by the addition of 30 µl kinase buffer containing 1 mM DTT, 5 µM unlabeled ATP, and 1 µCi of [{gamma}-32P]ATP (NEN, Cambridge, U.K.). Samples were incubated for 10 min at room temperature and the reaction stopped by the addition of 30 µl Laemmli’s buffer and boiling for 5 min. Radiolabeled proteins were resolved by 8% SDS-PAGE and transferred onto Immobilin-P. Dried membranes were exposed for autoradiography.

Confocal imaging

Isolated CD4+ T cells (4 x 107 cells) were prepared in one of two ways. 1) Cells were incubated for 10 min at 37°C in the presence of YHB.46 then washed once before stimulation with biotin-UCHT1 for 15 min at 37°C. The cell suspension was washed once before cross-linking with streptavidin conjugated to Texas Red for 5 min at 37°C. The cells were then washed and fixed with 1% paraformaldehyde in PBS containing 1% FCS and 0.01% sodium azide and left on ice for 5 min. The cell suspension was then incubated for 10 min at 4°C with an anti-human FITC IgG Ab to label the CD4. 2) Cells were incubated for 15 min at 37°C in the presence of biotin-UCHT1 for 15 min at 37°C. The cell suspension was washed once before cross-linking with streptavidin-Texas Red for 5 min at 37°C. The cells were then washed and fixed with 1% paraformaldehyde in PBS containing 1% FCS and 0.01% sodium azide and left on ice for 5 min. The cell suspension was then incubated for 10 min at 4°C with YHB.46 before washing and labeling with an anti-human FITC IgG Ab.

For confocal analysis, we embedded the fixed and stained cells in a 1% agarose solution in PBS and let them settle on coverslips ~5 min before the imaging. Spatially resolved localization of the labels was performed through a high numerical aperture (1.4) x100 objective on the stage of an Olympus IX 70 microscope (Olympus, Melville, NY) attached to an UltraView LCI (PerkinElmer Life Sciences, Cambridge, U.K.) laser scanning confocal microscope. Optical sections were <600 nm in thickness. FITC and Texas Red labels were excited with the 488- and 568-nm laser line of an attached Argon/Krypton-mixed gas laser. For the colocalization analysis, it was important to establish the level of cross-talk between the green (FITC) and red (Texas Red) channel in control experiments. The cross-talk was estimated to be <1%, and was thus neglected during our study. For each experimental condition, at least 10 cells were picked randomly.

The internalization of the TCR complex was analyzed on double-labeled cells by superimposing the FITC and Texas Red fluorescence images of the cells in a composite RGB image (ImageSuite 4.0, PerkinElmer LifeSciences). Color images obtained from three separate experiments were thereafter inspected by observers not involved in this study under "blind" conditions to count internalization events.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
YHB.46 mAb inhibits CD3-induced protein phosphorylation in primary human CD4+ T cells

Initial experiments were conducted to determine the optimal system for ligating the TCR expressed on highly purified primary human CD4+ T lymphocytes in the presence of YHB.46 CD4 mAb in such a way that the possibility of unwanted TCR-CD4 coligation was excluded. After comparing several systems, a standard protocol was established in which T cells were exposed to soluble YHB.46 for 10 min before TCR-stimulation using the UCHT1 CD3 mAb coated onto 0.1-µm latex beads. Fig. 1Goa illustrates an experiment using this protocol showing that whereas exposure of cells to soluble YHB.46 alone induced a modest increase in protein tyrosine phosphorylation (lane 2 cf lane 1), YHB.46 addition before TCR stimulation for 5 min caused a generalized decrease in the induction of tyrosine phosphorylation in multiple proteins (Fig. 1Goa, upper panel, arrows, lane 4 cf lane 3). Preincubation with a humanized mouse lysozyme isotype-matched 2G6 mAb as control had no effect on CD3-stimulated tyrosine phosphorylation (data not shown). Phosphorimager analysis revealed that the inhibition in CD3-stimulated phosphorylation events by YHB.46 varied in the range 23–81%, depending on which protein was measured, indicating a considerable degree of selectivity in the extent of inhibition observed. However, the extent of inhibition in specific proteins was comparable between donors.



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  		FIGURE 1. YHB.46 inhibits CD3-induced protein tyrosine phosphorylation in primary CD4+ T cells. a, CD4+ T cells were incubated with (lanes 2 and 4) or without (lanes 1 and 3) 20 µg/ml YHB.46 for 10 min before stimulation with (lanes 3 and 4) or without (lanes 1 and 2) UCHT1 (immobilized onto latex beads) for 5 min at 37°C. Cells were lysed and immunoprecipitated with Fb2 phosphotyrosine mAb. Proteins were separated by 10% SDS-PAGE and immunoblotted with phosphotyrosine mAb 4G10, followed by stripping and reprobing of the membrane with Vav, SLP-76, or p56lck Abs. Proteins displaying inhibition by YHB.46 mAb of CD3-stimulated tyrosine phosphorylation are indicated by arrows. The large arrowhead indicates the position of protein-G. Results are representative of four experiments using purified T cells from four different donors. b, CD4+ T cells from three separate donors were isolated and combined before incubation with (lanes 2, 4, 6, 8, and 10) or without (lanes 1, 3, 5, 7, and 9) 20 µg/ml YHB.46 for 10 min then stimulated with (lanes 3–10) or without UCHT1 (immobilized onto latex beads) for the time indicated at 37°C. Cells were lysed and immunoprecipitated with Fb2 (a) and the membrane probed with 4G10. Lane 11, A control in which protein-G Sepharose bound to Fb2 mAb was loaded on the gel without prior exposure to cell lysate. Results are representative of three separate experiments. c, ZAP-70 tyrosine phosphorylation (b) was quantified and results expressed as a percentage inhibition of control at each stimulation time point.

 
Many of the proteins that become tyrosine phosphorylated upon TCR ligation have been identified and include the p56lck tyrosine kinase, the TCR-{zeta} chain, the ZAP-70 tyrosine kinase, and adaptor proteins (26, 29). One of the adaptor molecules that has been extensively studied is SLP-76 (29). Following TCR activation, SLP-76 becomes tyrosine phosphorylated by ZAP-70 (54), and so generates binding sites for the Src homology 2 domains of various downstream effectors including Vav, the guanine nucleotide exchange factor for the Rac/Rho family of GTPases (28, 55). Preincubation with the CD4 mAb strongly reduced the tyrosine phosphorylation of a 76-kDa protein (Fig. 1Goa). Western blotting of the same membrane with SLP-76 mAb showed that this phosphotyrosine containing protein coincided with SLP-76 and that the amount of SLP-76 protein precipitated by the phosphotyrosine mAb was reduced (by 45%) after treatment with YHB.46 (Fig. 1Goa, lower panel), confirming that CD3-stimulated phosphorylation of SLP-76 was inhibited by CD4 ligation. A protein migrating at 95 kDa, which likewise displayed reduced phosphorylation following treatment of cells with YHB.46, was identified as Vav by immunoblotting (Fig. 1Goa, lower middle panel). Reprobing for p56lck (Fig. 1Goa, bottom panel) identified the 56-kDa phosphorylated protein (arrowed, upper panel, lane 3) as p56lck, suggesting that the CD3-stimulated increase in p56lck phosphorylation was also reduced by the YHB.46 mAb treatment (by 61% as measured by phosphorimager analysis of the upper panel). It should be noted that the apparent increase in p56lck protein in the phosphotyrosine immunoprecipitates, illustrated in lanes 2 and 4 (Fig. 1Goa, bottom panel), was most likely due to the binding of a small amount of YHB.46 mAb, incompletely removed by preclearing, to the protein-G Sepharose during the immunoprecipitation process. Because CD4 binds p56lck very efficiently, even a small amount of CD4 mAb is expected to coprecipitate a significant amount of p56lck. Irrespective of this interpretation, the upper panel of Fig. 1Goa clearly shows that CD3-stimulated p56lck phosphorylation was reduced by prior exposure of the cells to YHB.46.

We considered that the inhibitory effects of preincubating YHB.46 with T cells before CD3 stimulation might vary depending on the length of time that the TCR was engaged with the CD3 mAb. To investigate this question, the inhibition of ZAP-70 phosphorylation was measured over a 2–20-min time course by immunoprecipitating tyrosine-phosphorylated proteins. Fig. 1Gob shows that the phosphorylation of a protein migrating at 70 kDa was inhibited over this time course by YHB.46 preincubation. The protein was identified by immunoblotting which also revealed that the amount of ZAP-70 protein found in the immunoprecipitates was reduced by YHB.46, consistent with the reduction in tyrosine phosphorylation (Fig. 1Gob, lower panel). Interestingly, phosphorimager analysis of ZAP-70 phosphorylation (Fig. 1Goc) revealed that the extent of inhibition by YHB.46 (30–40%) was comparable over the entire CD3 stimulation time investigated. Therefore, prior exposure of T cells to YHB.46 is effective in inhibiting TCR-stimulated phosphorylation events over a prolonged time period.

We also considered that the results illustrated in Fig. 1Goa might be misleading in the event that the immunoprecipitating phosphotyrosine Fb2 mAb did not gain effective access to phosphotyrosine residues in nondenatured proteins. To assess this concern, we chose Vav as a test protein, as this signaling molecule is known to contain several CD3-stimulated tyrosine phosphorylation sites (56). Vav was immunoprecipitated directly from cells treated as in Fig. 1Goa and then immunoblotted for phosphotyrosine. As Fig. 2Goa illustrates, the inhibition (by 65%) of CD3-stimulated Vav phosphorylation by YHB.46 observed using this method (lane 4 cf lane 3) was comparable to that obtained using the method shown in Fig. 1Goa. Stripping and reprobing of the membrane demonstrated that the amount of Vav protein present in each immunoprecipitate was comparable (Fig. 2Goa, lower panel). We conclude from this experiment that immunoprecipitation of tyrosine-phosphorylated proteins using the Fb2 mAb provides a reliable way of assessing the level of CD3-stimulated phosphorylation events, and that the inhibition by YHB.46 can be independently confirmed by immunoprecipitating specific proteins.



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  		FIGURE 2. YHB.46 inhibits CD3-induced protein tyrosine phosphorylation of the adapter proteins Vav and LAT in primary CD4+ T cells. CD4+ T cells from separate donors were treated as in Fig. 1Goa and either Vav (a) or LAT (b) immunoprecipitated from whole-cell lysates before immunoblotting for phosphotyrosine (upper panel) or, by stripping and reprobing, for the immunoprecipitated protein (lower panel). Lane 5, A control in which protein-G Sepharose bound to the immunoprecipitating Ab was loaded on the gel without prior exposure to cell lysate. Results are representative of three experiments using purified T cells from three different donors.

 
Another important adapter protein lying downstream of ZAP-70 in T cell activation is LAT (57). This molecule is also of interest as it has been reported to associate with CD4 (58). Preincubation with the CD4 mAb strongly reduced the tyrosine phosphorylation of a protein migrating in the 36–38-kDa region that appeared to be LAT (Fig. 1Goa). To address this question more directly, LAT was immunoprecipitated from cells treated as in Fig. 1Goa and then immunoblotted for phosphotyrosine. Fig. 2Gob shows that YHB.46 inhibits the CD3-stimulated phosphorylation of LAT (by 44%). Reprobing of the membrane with LAT Ab demonstrated that the amount of LAT protein present in each lane was comparable (Fig. 2Gob, lower panel). For technical reasons due to the use of a CD4 mAb as the inhibitory reagent, we were unable to investigate whether the putative CD4-associated pool of LAT was more or less inhibited than the total pool of LAT molecules as measured in Fig. 2Gob.

Overall, these results show that preexposure of cells to YHB.46 mAb causes the inhibition of multiple CD3-stimulated phosphorylation events, and that the proteins involved include those already known to be key mediators of downstream signaling pathways involving the formation of signal transduction complexes and transcription factor regulation.

CD4 mAb inhibits TCR coupling to the Ras/MAPK pathway in primary human CD4+ T cells

The YHB.46-mediated inhibition of tyrosine phosphorylation events illustrated in Figs. 1Go and 2Go was never complete in any of the T cell preparations from the multiple donors used for these assays. Therefore, we wished to confirm that the levels of inhibition attained by YHB.46 had measurable effects on downstream signals known to be important in T cell activation. In T cells, p21ras couples receptor-stimulated protein tyrosine kinases via Raf-1 to the MAPK cascade (32). The effects of YHB.46 on this cascade were investigated by immunoblotting whole-cell lysates from unstimulated or CD3-activated cells with an Ab to the phosphorylated form of p42/44 MAPK (Fig. 3Go, upper panel). Preincubation of the cells with YHB.46 led to a reduction in phosphorylation of 42-kDa (72% inhibition) and 44-kDa (66% inhibition) proteins in CD3-stimulated cell lysates. The proteins were identified as the Erk1 and Erk2 members of the MAPK family by reprobing the membrane using specific Abs (Fig. 3Go, lower panels). These results confirm that the levels of inhibition of signaling protein phosphorylation achieved by YHB.46 (Figs. 1Go and 2Go) are sufficient to achieve a significant reduction in the activation of an important downstream signaling cascade.



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  		FIGURE 3. YHB.46 inhibits CD3-induced Erk1/2 activation. CD4+ T cells were incubated with (lanes 2 and 4) or without 20 µg/ml YHB.46 (lanes 1 and 3) for 10 min before stimulation with (lanes 3 and 4) or without (lanes 1 and 2) UCHT1 (immobilized onto latex beads) for 5 min at 37°C. Whole-cell lysates were separated by 10% SDS-PAGE and visualized by immunoblotting with phospho-42/44 MAPK mAb. The blot was then stripped and reprobed with Erk1 and then Erk2 mAbs. Results are representative of four different experiments using T cell preparations from different donors.

 
YHB.46 inhibits the earliest initiation steps in TCR signal transduction coupling

The finding that YHB.46 caused a general inhibition in the phosphorylation of multiple proteins (Fig. 1Go) suggested that its inhibitory locus of action was likely to be very early in the TCR signaling cascade. Tyrosine phosphorylation of the TCR-{zeta} chain is critical for the initiation of signal transduction, resulting in the recruitment of ZAP-70 and the amplification of downstream signals (17). Therefore, the effect of YHB.46 on TCR-{zeta} chain phosphorylation and ZAP-70 recruitment was investigated in TCR-{zeta} immunoprecipitates. Fig. 4Goa shows that CD3 stimulation caused, as expected, an increase in the p23 phosphoisomer of the TCR-{zeta} chain and led to recruitment and phosphorylation of a 70-kDa protein (upper panel) identified as ZAP-70 by reprobing the blot (middle panel). Preincubation with YHB.46 resulted in a reduction of 36 and 25%, respectively, in the generation of the TCR-{zeta} p21 and p23 phosphoisomers (upper panel), a reduction also observed by immunoblotting the precipitates with a TCR-{zeta} Ab (lower panel). This was associated with a 49% reduction in the recruitment of ZAP-70 to the TCR-{zeta} phosphoisomers (middle panel, lane 4, cf lane 3). Fig. 4Gob illustrates a separate experiment using purified T cells from a different donor showing that the recruitment of ZAP-70 to TCR-{zeta} in this case was reduced by 58% following addition of YHB.46.



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  		FIGURE 4. YHB.46 inhibits CD3-induced tyrosine phosphorylation of the TCR-{zeta} chain, and recruitment and phosphorylation of ZAP-70. CD4+ T cells were incubated with (lanes 2 and 4) or without 20 µg/ml YHB.46 (lanes 1 and 3) for 10 min before stimulation with (lanes 3 and 4) or without (lanes 1 and 2) UCHT1 (immobilized onto latex beads) for 5 min at 37°C. Cells were lysed and immunoprecipitated with goat anti-{zeta} N39 (CT-11) antiserum (a–c), with rabbit polyclonal ZAP-70 antiserum (d), or with rabbit polyclonal phospho-Tyr319-ZAP-70 antiserum (e). Immunoprecipitated proteins were separated by 7–15% gradient SDS-PAGE (a–d) and 8% (e) SDS-PAGE. a, The TCR-{zeta} immunoprecipitate was probed for phosphotyrosine (upper panel), and then stripped and reprobed for ZAP-70 (middle panel) and TCR-{zeta} (lower panel). b, CD4+ T cells from a separate donor were treated exactly as in a. c, The TCR-{zeta} immunoprecipitate was subjected to an in vitro kinase assay (upper panel) before immunoblotting for p56lck (lower panel). Note the protein shown as ZAP-70 was identified by reprobing the membrane with a ZAP-70 mAb (data not shown). d, The ZAP-70 immunoprecipitates were probed for phosphotyrosine (upper panel) followed by stripping and reprobing of the membrane for ZAP-70 (middle panel) and TCR-{zeta} (lower panel). The large arrowhead indicates the position of protein-G. e, The phospho-ZAP-70 immunoprecipitates were probed for phospho-ZAP-70 (upper panel) followed by stripping and reprobing of the membrane for ZAP-70 (lower panel). a, d, and e, lane 5, A control in which protein-G Sepharose bound to the immunoprecipitating Ab was loaded on the gel without prior exposure to cell lysate. The results are representative of four separate experiments using T cell preparations from four different donors.

 
The actions of YHB.46 were further elucidated by carrying out in vitro kinase assays in the TCR-{zeta} immunocomplex, as illustrated in Fig. 4Goc. The recruitment and phosphorylation of a prominent 56-kDa protein, identified as p56lck by immunoblotting (lower panel), was markedly increased by CD3-stimulation, in parallel with the generation of multiple TCR-{zeta} phosphoisomers (upper panel). A modest increase in phosphorylation of a protein identified by immunoblotting as ZAP-70 (arrowed) was also observed. The increased recruitment of p56lck protein to TCR-{zeta} upon CD3-stimulation (lower panel, lane 3, cf lane 1), together with increased ZAP-70 recruitment (Fig. 4Go, a and b), is consistent with the previously reported association of p56lck with ZAP-70 following T cell activation (23, 24, 59, 60). Upon preincubation with YHB.46, the subsequent recruitment to TCR-{zeta}/ZAP-70 and phosphorylation of p56lck was reduced by 50%, and the in vitro phosphorylation of the TCR-{zeta} chain p56lck substrate was likewise reduced. The most straightforward interpretation of these findings is that YHB.46 reduces the phosphorylation of TCR-{zeta} with a concomitant reduction in ZAP-70 recruitment (as in Fig. 4Go, a and b); in turn, less p56lck is recruited into the TCR-{zeta} complex (Fig. 4Goc) since less ZAP-70 is available for binding p56lck (60).

The effects of YHB.46 on ZAP-70 were further investigated by immunoblotting ZAP-70 immunoprecipitates. Fig. 4God shows that YHB.46 markedly reduced (72% inhibition) the CD3-stimulated increase in tyrosine phosphorylation of a protein (upper panel, lane 4, cf lane 3) identified as ZAP-70 by reprobing the membrane (middle panel). At the same time there was also a striking inhibition (by 50%) in the production of a p23 protein identified as the p23 TCR-{zeta} phosphoisomer by reprobing using specific Ab (lower panel). Therefore, the main phosphoisomer of TCR-{zeta} to associate with ZAP-70 in stimulated cells was the p23 phosphoisomer, which is known to be phosphorylated in all three ITAM motifs (61); using this assay it was selectively this p23 phosphoisomer that was inhibited by YHB.46. In comparing these results with those illustrated in Fig. 4Goa, it should be noted that immunoblotting of TCR-{zeta} immunoprecipitates for phosphotyrosine reveals the tyrosine phosphorylation level of the total detergent-soluble pool of TCR-{zeta} polypeptides, whereas probing ZAP-70 immunoprecipitates reveals the phosphorylation status of the particular TCR-{zeta} pool to which ZAP-70 predominantly binds. The effect of YHB.46 on the tyrosine phosphorylation state of ZAP-70 was further investigated by immunoprecipitating with a phospho-ZAP-70 Ab, which detects ZAP-70 only when it is phosphorylated at Tyr319 within the linker region of the molecule. Phosphorylation at this site is thought to be responsible for the binding of p56lck (23, 24), and is important in transducing TCR signals to downstream pathways such as the Ras/MAPK and calcium signaling pathways. Fig. 4Goe shows that YHB.46 markedly reduced (67% inhibition) the CD3-stimulated increase in tyrosine phosphorylation of immunoprecipitated pTyr319-ZAP-70 (upper panel, lane 4 cf lane 3) confirmed as ZAP-70 by reprobing the membrane (lower panel).

Overall, these results suggest that YHB.46 acts very early in TCR signal transduction coupling to reduce the phosphorylation of the TCR-{zeta} chain, with a consequent reduction in ZAP-70 recruitment and tyrosine phosphorylation, especially at Tyr319, in turn reducing p56lck binding to ZAP-70.

YHB.46 inhibits TCR internalization in primary human CD4+ T cells

It has been suggested that inhibition of TCR signaling by CD4 ligation could be due to sequestration of p56lck away from the TCR complex, thereby preventing the actions of p56lck in phosphorylating TCR-{zeta} and ZAP-70 (46, 47, 48, 62), although other findings are inconsistent with this model (49, 50). Therefore, we treated primary resting CD4+ T cells with UCHT1 and YHB.46 mAbs and visualized the fluorescence labeling of CD3 and CD4 by confocal microscopy to elucidate CD4 localization in relation to the TCR. Cells were preincubated either with or without YHB.46 before CD3 stimulation. Fig. 5Goa shows that exposure of T cells to soluble YHB.46 for 10 min at 37°C, before CD3 stimulation, the same protocol used for all the signaling data illustrated in Figs. 1–4GoGoGoGo, caused no obvious patching, capping, or detectable internalization of CD4 molecules. Visualization of UCHT1-Texas Red staining of TCR molecules in the same cell (illustrated in Fig. 5Goc) showed that CD3 stimulation, as expected, resulted in TCR capping. When the CD3 stimulation was conducted without prior exposure to YHB.46 (as shown in Fig. 5Gob), the internalization was particularly marked during the 5 min of CD3 cross-linking used for stimulation. Staining of the same cell following fixing with YHB.46-FITC (data not shown) revealed a uniform staining of CD4 at the cell surface, as in Fig. 5Goa. Interestingly, as Fig. 5Goc illustrates, CD3-stimulated TCR internalization was much reduced (by 58%, taking the average results from 15 cells analyzed from three separate donors) following preincubation of cells with YHB.46. Given that TCR internalization is thought to be important in the serial engagement model of T cell activation (33, 34), these results revealed a further significant way in which the CD4 mAb may lower the intensity of TCR signaling. However, our experiments provided no evidence for p56lck sequestration away from the TCR, although such effects below the level of detection of confocal imaging cannot be excluded.



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  		FIGURE 5. YHB.46 inhibits CD3-induced internalization of the TCR. a, YHB.46-FITC-labeled and UCHT1-Texas Red-labeled CD4+ T cell. The figure shows YHB.46-FITC (CD4) labeling only viewed at 488 nm. CD4+ T cells (4 x 107 cells) were incubated for 10 min at 37°C with 20 _g/ml YHB.46 before stimulation with 5 µg/ml biotin-UCHT1 for 15 min at 37°C followed by cross-linking with streptavidin-Texas Red for 5 min at 37°C. The cells were then fixed with 1% paraformaldehyde before labeling with an anti-human FITC IgG Ab for 10 min at 4°C to reveal the CD4 staining by YHB.46. b, UCHT1-Texas Red and YHB.46-FITC labeled CD4+ T cell. The figure shows Texas Red (CD3) staining measured at 568 nm. CD4+ T cells (4 x 107 cells) were incubated for 15 min at 37°C with biotin-UCHT1 for 15 min at 37°C before cross-linking with streptavidin-Texas Red for 5 min at 37°C. The cells were then fixed with 1% paraformaldehyde before labeling with YHB.46 by incubating for 10 min at 4°C and a further 10 min in the presence of an anti-human FITC IgG Ab. c, YHB.46-FITC-labeled and UCHT1-Texas Red-labeled CD4+ T cell. The figure shows Texas Red (CD3) staining measured at 568 nm. CD4+ T cells were prepared as in a.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main findings in this paper are that a humanized CD4 mAb, YHB.46, inhibits the earliest initiation events of TCR signal transduction, such as TCR-{zeta} phosphorylation and ZAP-70 recruitment and phosphorylation at Tyr319 (Fig. 4Go), leading to a reduction in TCR internalization (Fig. 5Go), a general inhibition of the multiple protein tyrosine phosphorylation events arising from TCR stimulation (Figs. 1Go and 2Go) and inhibition of the Ras/MAPK pathway (Fig. 3Go).

It seems likely that the previously reported inhibition by CD4 ligation of downstream signaling pathways may also have been due to the actions of CD4 mAbs on the initial TCR coupling events. For example, inhibition of the formation of signal transduction complexes, such as those formed between p120GAP, phospholipase C-{gamma}1 and other molecules (40), calcium signals (41, 42), Erk-2 and c-Jun N-terminal kinase activation (43, 44, 63), and transcription factor activation (44, 45) would all be expected in the event that TCR-{zeta} phosphorylation and ZAP-70 recruitment were inhibited. The present work extends previous reports on the inhibition of signaling complexes by showing that TCR-induced Vav (Fig. 2Go) and SLP-76 (Fig. 1Go) phosphorylation are also inhibited by a CD4 mAb. Interestingly, it has been reported that in the context of murine CD4+ T cell clones, TCR stimulation by an agonist peptide in the presence of a CD4 mAb caused preferential generation of the p21 TCR-{zeta} phosphoisomer rather than the fully phosphorylated p23 phosphoisomer, with a striking reduction in ZAP-70 phosphorylation. This TCR signaling profile was similar to that induced using an altered peptide ligand (46). In the present work, YHB.46 likewise caused a marked inhibition in generation of the specific p23 TCR-{zeta} phosphoisomer pool associated with ZAP-70 (Fig. 4God) and a large reduction in ZAP-70 tyrosine phosphorylation (Fig. 4Go, a, d, and e). Therefore, our results demonstrate that a CD4 mAb can down-regulate the earliest initiation events of Ag receptor phosphorylation in primary resting human CD4+ T cells, and that such molecular mechanisms of inhibition are not restricted to preactivated T cell clones that require maintenance by regular cycles of Ag stimulation.

The p56lck tyrosine kinase is known to play important roles in mediating phosphorylation of the TCR-{zeta} chain (11) and ZAP-70 (11, 64), and the degree of p56lck binding to ZAP-70 is in turn dependent on the level of ZAP-70 phosphorylation (23, 24, 59, 60). The actions of p56lck are regulated by the CD45 tyrosine phosphatase (22). In CD45-/- thymocytes, in which p56lck is dysfunctional, CD3-stimulated TCR-{zeta} tyrosine phosphorylation and ZAP-70 recruitment and phosphorylation are severely reduced (20) in a manner similar to that evoked by YHB.46 in the present work (Fig. 4Go). We have also recently presented evidence that engagement of CTLA-4 causes a marked reduction in CD3-stimulated phosphorylation of ZAP-70-Tyr319 with a consequent reduction in p56lck association pTyr319 (65), so YHB.46 also appears to mimic, by a different mechanism, some of the effects of CTLA-4 ligation in primary resting human T cells. In fact, the actions of YHB.46 can most readily be explained by a sequestration model in which binding of YHB.46 to CD4 molecules perturbs the ability of CD4/p56lck to be used by the TCR following ligation of the receptor. In a variant of this model, the CD4 mAb might prevent associations between CD45 and CD4 that are important for CD45-mediated regulation of the CD4-associated pool of p56lck (66, 67, 68). Assays of p56lck kinase activity in p56lck immunoprecipitates from CD3-stimulated cells with or without prior incubation with YHB.46 did not reveal any change in kinase activity (data not shown). Physical prevention of p56lck from effective engagement with its substrates, rather than kinase inhibition, would readily explain its inability to phosphorylate TCR-{zeta} and ZAP-70, and might also contribute to the reduced recruitment of p56lck to the TCR-{zeta}/ZAP-70 complex (Fig. 4Goc), although this could just as well be explained by the reduction in ZAP-70 phosphorylation at Tyr319 (Fig. 4Goe). How the YHB.46 might prevent the accessibility of CD4/p56lck to TCR/ZAP-70 is less clear. Our confocal analysis provided no evidence for patching or capping of CD4 molecules following binding of soluble YHB.46 to cells (Fig. 5Go), a finding that might have provided evidence for a physical separation between surface pools of CD4 and TCR molecules (which do cap, Fig. 5Gob). However, it is possible that simple dimerization of CD4 by soluble YHB.46 mAb might lower its lateral mobility within the lipid bilayer, rendering CD4-associated p56lck less available for TCR/ZAP-70 phosphorylation.

Sequestration models have been previously evoked to explain the inhibition of TCR signaling when p56lck is bound to CD4 without CD4-TCR ligation (48), the down-regulation of TCR signaling upon HIV gp120-mediated translocation of p56lck to the cytoskeletal fraction (62), and the hyporesponsiveness of thymocytes from nonobese diabetic mice (69). In contrast, p56lck-independent inhibitory effects of CD4 mAbs have been demonstrated by reconstituting T cell clones or hybridomas with CD4 mutants unable to bind p56lck (49, 50). Therefore, it cannot be excluded that other mechanisms may be involved in the mediation of negative signals by CD4 mAbs. For example, the adaptor molecules LAT (58) and SLP-76 (70) have been reported to associate with CD4 and p56lck, respectively, and so CD4 ligation could potentially disrupt TCR-mediated signaling via these molecules. In addition, a novel CD4-interacting protein, acidic cluster protein 33, has recently been identified as a candidate molecule for mediating negative signaling by the hydrophobic C-terminal of CD4 (71). Nevertheless, in the present work the YHB.46-mediated reduction in tyrosine phosphorylation of adaptors such as LAT (Fig. 2Gob), SLP-76 (Fig. 1Go), and Vav (Fig. 2Goa) are events that lie downstream of the inhibition in TCR-{zeta} phosphorylation and ZAP-70 recruitment (27, 28, 29), so a direct perturbation of p56lck actions is sufficient to explain the subsequent inhibition of all these downstream signals.

An important finding from our confocal studies was that exposure of CD4+ T cells to YHB.46 inhibited the subsequent internalization of CD3-bound TCR molecules by 58% (Fig. 5Goc), a level of inhibition comparable with the inhibition of TCR-{zeta}/ZAP-70 phosphorylation (Fig. 4Go). Following exposure of T cells to Ag or to stimulating CD3 mAbs, TCR molecules are rapidly internalized (72, 73, 74). Internalization domains that include ITAM motifs have been described in the CD3 polypeptides (75, 76, 77). However, whether tyrosine phosphorylation of the TCR is necessary for endocytosis remains unresolved. Irrespective of the precise mechanism whereby YHB.46 retards TCR internalization, if this inhibitory process also applies to the internalization of MHC-peptide-stimulated receptors in vivo then, given the potential importance of serial engagement in TCR signaling (33, 34), this may represent a further important mechanism whereby CD4 mAbs can suppress T cell activation in vivo.

Overall, our findings show that the YHB.46 mAb is a powerful inhibitor of the early initiating events of T cell signaling.


   Acknowledgments
 
We thank Prof. Hill Gaston for reviewing an earlier version of the paper, and we thank the following for their kind provision of reagents: Prof. P. Beverley, Prof. D. Cantrell, Dr. R. Field, Dr. G. Hale, Dr. G. Koretzky, Dr. L. Samelson, Dr. M. Turner, Prof. C. Terhorst, Janssen-CILAG, and GlaxoSmithKline.


   Footnotes
 
1 Supported by the Arthritis Rheumatism Council and the Biotechnology and Biological Sciences Research Council Back

2 Address correspondence and reprint requests to Dr. Denis R. Alexander, Laboratory of Lymphocyte Signaling and Development, Molecular Immunology Program, The Babraham Institute, Cambridge CB2 4AT, U.K. E-mail address: denis.alexander{at}bbsrc.ac.uk Back

3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; Erk, extracellular signal-related kinase; LAT, linker for activation of T cells; SLP-76, Src homology 2 domain-containing leukocyte protein of 76 kDa; ZAP-70, {zeta}-associated protein of 70 kDa; Vav, p95vav. Back

Received for publication August 8, 2001. Accepted for publication April 19, 2002.


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 Materials and Methods
 Results
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