CD4 expressed on transfected HeLa and NIH3T3 cells was internalized. Approximately 1.5-2% of the total cell- bound [1251]Fab' fragments were internalized ...
The EMBO Journal vol.8 no.12 pp.3641 -3649, 1989
Internalization and recycling of CD4 transfected into HeLa and NIH3T3 cells
Annegret Pelchen-Matthews, Jane E.Armes and Mark Marsh Chester Beatty Laboratories, Institute of Cancer Research, Fulham Road, London SW3 6JB, UK Communicated by R.A.Weiss
The internalization of CD4, a T cell differentiation antigen and the receptor for the human immunodeficiency viruses (HIV-1 and -2), has been examined in HeLa and murine 3T3 cells transfected with CD4 cDNA. Fab' fragments of the anti-CD4 monoclonal antibody Leu3a were generated by pepsin digestion and used as a specific monovalent, non-crosslinking ligand for CD4. These Fab' fragments were shown to bind to CD4 on the transfected cells with an affinity similar to that of HIV gpl20, and inhibited HIV infection of lymphocytic cells. The Fab' fragments were radioiodinated and used in an acid-stripping endocytosis assay to demonstrate that the CD4 expressed on transfected HeLa and NIH3T3 cells was internalized. Approximately 1.5-2% of the total cellbound [1251]Fab' fragments were internalized per minute. Furthermore, the internalized ['251]Fab' fragments could be shown to recycle to the cell surface. After 30-60 min a steady state was reached between internalization and recycling, with 30-40% of the total cellular CD4 pool residing inside the cell. Similar results were obtained in studies with the intact divalent radiolabelled Leu3a antibody. These data demonstrate that CD4 expressed on transfected non-lymphoid cells is constitutively endocytosed and recycled. Key words: CD4/endocytosis/recycling/virus receptors -
Introduction CD4 is a differentiation antigen expressed primarily on T lymphocytes which interact with class II major histocompatibility complex (MHC)-bearing cells, and on some cells of the macrophage/monocyte lineage. The CD4 molecule is a transmembrane glycoprotein of 55 kd mol. wt containing four extracellular immunoglobulin-like domains (Maddon et al., 1985), thus warranting its inclusion in the immunoglobulin gene superfamily. Although the expression of cell surface CD4 defines certain T cell subsets, the precise role of the molecule in T cell function is unknown. Anti-CD4 antibodies have been shown to inhibit antigen-stimulated T cell activation (reviewed by Parnes, 1989), suggesting that CD4 may modulate the activity of the T cell receptor (TCR). It has been proposed that CD4 may augment low-affinity -
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interactions between the TCR and antigen-presenting cells by binding to monomorphic determinants on class II MHC antigens (Doyle and Strominger, 1987; Gay et al., 1987; Sleckman et al., 1987). In addition there is some evidence that CD4 is itself capable of transmembrane signal transduction (Bank and Chess, 1985; Wassmer et al., 1985; Tite et al., 1986; Rosoff et al., 1987; Carrel et al., 1988), possibly in concert with a lymphocyte-specific protein tyrosine kinase (Veillette et al., 1988, 1989; Rudd et al., 1988), and that it may function as a growth factor receptor (Cruikshank et al., 1987; Kornfeld et al., 1988). CD4 is known to act as the receptor for the human immunodeficiency viruses (HIV-1 and -2) and in this respect it has become the best characterized of animal virus receptors (Dalgleish et al., 1984; Klatzmann et al., 1984; McDougal et al., 1986; Maddon et al., 1986). Despite its demonstrated role in T cell activation, very little is known about the biology of CD4. The cell surface expression of CD4 has been shown to be downregulated following treatment of cells with phorbol esters (Acres et al., 1986; Hoxie et al., 1986, 1988; Clapham et al., 1987) or during antigenic stimulation (Blue et al., 1987; Weyand et al., 1987; Rivas et al., 1988). This modulation appears to involve the phosphorylation of serine residues in the cytoplasmic domain of the molecule (Acres et al., 1986). It has also been demonstrated that CD4-positive cells can present HIV gpl2O envelope protein antigens to primed T cells (Lanzavecchia et al., 1988; Siliciano et al., 1988, 1989). All these activities of CD4 presumably require the internalization of the molecule. However, to date very little is known about the endocytosis of CD4. In part, at least, this may be attributed to difficulties in studying endocytosis in lymphoid cells. The molecular cloning of CD4 and transfection of cDNA into non-lymphoid cells has allowed the expression of CD4 on the surface of a variety of adherent tissue culture cells that are more conducive to the analysis of endocytosis. While it is unclear how the behaviour of the molecule on these cells reflects that of CD4 on T cells, the transfected molecule is functional to the extent that CD4-negative human cells, when transfected with CD4 cDNA, become susceptible to infection by HIV (Maddon et al., 1986, 1988). The availability of the CD4-transfected cells has enabled assays for the endocytosis of the molecule to be established, which may subsequently be applied to T cell systems. We have used monovalent radiolabelled Fab' fragments of the anti-CD4 monoclonal antibody Leu3a to determine biochemically the cellular distribution of CD4 in HeLa and NIH3T3 cells expressing transfected CD4. We demonstrate that CD4 is constitutively internalized and recycled in these cells and that there is essentially no difference in the behaviour of the molecule when expressed in cells of human and murine origin.
3641
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Fig. 1. Fab' fragments of Leu3a. Analysis of Leu3a IgG (lane 1), a reduced and alkylated pepsin digest of Leu3a (lane 2) and purified Fab' fragments of Leu3a (lane 3) by reducing SDS-PAGE. The positions of mol. wt standards (in kd) are indicated. HC, immunoglobulin heavy chain; LC, immunoglobulin light chain; HCF, pepsin digestion fragment of the immunoglobulin heavy chain.
Table I. Fab' fragments of Leu3a inhibit syncytium formation on C8166 cells
1251-Fab' froe (nM)
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Antibody
Dilution of antibody stock control 1/50 1/10
Leu3a (20 1g/ml, 0.13 tM) Fab' of Leu3a (60 tg/ml, 1.1 oM) Phosphate buffer
-
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+ + + + ++
+++ ++ +++
C8166 cells were incubated overnight with antibody and 105 TCID50 units of HIV-1/well in a 96 well plate and syncytium formation assessed after 24 h. Results ranged from absence of syncytia (-) to > 80% of cells in syncytia (+ ++).
Results Preparation of biologically active Fab' fragments of Leu3a Digestion of the anti-CD4 monoclonal antibody Leu3a with pepsin as detailed by Lamoyi and Nisonoff (1983) yielded a F(ab')2 preparation, which could be reduced to Fab' fragments with cysteine and alkylated using iodoacetamide. When the reduced and alkylated digest was fractionated by gel permeation HPLC, - 70% of the protein loaded was recovered as Fab'. The Fab' preparation had an apparent Mr of 47 000 on non-reducing SDS-PAGE and, on reduction, was shown to consist of the immunoglobulin light chain (Mr 28 000) and a fragment of the heavy chain (Mr 27 000; Figure 1, lane 3). The purified Fab' preparation was completely free of the immunoglobulin heavy chain present in the unpurified digest (Figure 1B, lane 2). The Leu3a Fab' fragments retained the ability to bind CD4 as shown by indirect immunofluorescence on HeLa or NIH3T3 cells transfected with human CD4 cDNA. The rhodamine-labelled goat anti-mouse immunoglobulin second antibody stained cells incubated with Fab' as strongly as cells treated with the native Leu3a antibody. No staining was observed with a non-specific antibody of the same sub-class
3642
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Fig. 2. Binding of [125I]Fab' fragments of Leu3a to HeLa-CD4.2G3 cells. (A) Time course of binding of [125I]Fab' fragments to CD4. HeLa-CD4 (0) or HeLa cells (0) were incubated in 4.5 nM [125I]Fab' fragments at 4°C. (B) Concentration dependence of binding of [125I]Fab' fragments to HeLa-CD4 (0) or HeLa cells (0). Cells were labelled at 4°C (2 h) in various concentrations of [125I]Fab' fragments. The inset shows a Scatchard plot of the specific binding, calculated by subtracting the non-specific binding to HeLa cells from the total binding on HeLa-CD4 cells. (C) Inhibition of binding of [125I]Fab' fragments on HeLa-CD4 cells by unlabelled Leu3a. Cells were incubated in 1.0 nM [125I]Fab' in the presence of various concentrations of Leu3a IgG for 2 h at 4°C. as Leu3a or on the CD4-negative parent cell lines, HeLa and NIH3T3, incubated with Leu3a Fab' (not shown). The biological activity of the Leu3a Fab' fragments was demonstrated by their ability to block HIV infection in culture. Incubation of C8166 cells with Fab' fragments (110 nM) prior to exposure to HIV completely inhibited the subsequent formation of syncytia (Table I). The Fab' fragments were not, however, as potent as the native Leu3a antibody in blocking HIV infection, presumably due to the reduced affinity of the monovalent fragments compared to the divalent IgG (see below).
Interaction of radioiodinated Fab' fragments or Leu3a with CD4 Fab' fragments of Leu3a were radioiodinated to high specific activity, diluted in ice-cold binding medium and used to label
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cells which were kept on ice (see Materials and methods). The [ 1251]Fab' preparation bound specifically to CD4-positive transfectants of HeLa or NIH3T3 cells, but showed negligible binding to the untransfected parent cells. An association binding time course on the CD4-positive HeLa clone 2G3 (Figure 2A) indicated that [1 51]Fab' binding reached saturation in 2 h. A study of the concentration dependence of binding and Scatchard analysis (Figure 2B) revealed an affinity of the interaction (Kd) of 10-9 M, with a maximal binding capacity (Bmax) of 3 650 fmol/mg total cell protein. Since the Fab' fragments were derived from a monoclonal antibody and therefore interact with CD4 with 1:1 stoichiometry, the Bmax corresponds to 200 000 molecules of CD4 per cell. The binding of [125I]Fab' was specifically inhibited by unlabelled native Leu3a (Figure 2C). The observed IC50 of 1 nM represents an apparent inhibition constant (Klapp) of 7.7 x 10-1° M for Leu3a binding; this is significantly higher than the affinity of the Fab' fragments, presumably as a result of the divalent binding of the intact antibody. Bound [125I]Fab' fragments could be removed from the cells by incubation with ice-cold acidified binding medium. Treatment at pH 4.5 or 4.0 for 10 min removed a significant proportion (63 and 86% respectively) of the bound [I 25]Fab' (Figure 3) while > 95 % of the bound [1251]Fab' was lost in 2 min at pH 3.0 (Figure 3, inset). The cold acid wash at pH 3 did not significantly affect the endocytic activity or viability of the cells since endocytosis of the fluid phase marker horseradish peroxidase, the incorporation of [35S]methionine into proteins, and the efficiency of replating was the same as that for untreated control cells (data not shown). Cell surface [1251]Fab' was therefore routinely removed using a 2 min wash on ice in pH 3 medium. As with the ['251]Fab' fragments, saturable binding of intact radioiodinated Leu3a ([ 1I]Leu3a) was observed only to CD4-positive cells. Scatchard analysis of the specific [1251]Leu3a binding to the HeLa-CD4.2G3 clone revealed x
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Fig. 3. Elution of bound [1251]Fab' fragments by media of different pH. Cells were labelled with 0.5 nM [125I]Fab' fragments of Leu3a at 4°C (2 h), washed and incubated (10 min at 4°C) in BM adjusted to the pH values indicated. The amount of label remaining on the cells is shown as a proportion of the binding to cells treated with BM, pH 7.3. The inset shows the time course of dissociation of [125 ]Fab' fragments at pH 3 ( 0 ) and pH 4 (0).
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Fig. 4. Internalization of [1251]Fab' fragments of Leu3a bound to CD4 on HeLa-CD4 cells. Cells were labelled with 0.5 nM [1251]Fab' fragments at 4°C (2 h), washed and warmed to 37°C for various times. Total cell-associated [125I]Fab' (0) and low-pH-resistant [1251]Fab' (0) were then determined as described in Materials and methods. The inset shows the low pH-resistant activity as a proportion of the total cell-associated [125I]Fab'. Error bars indicate the standard deviation of measurements from four culture wells.
Kd of 4.4 x 10-10 M, in agreement with the inhibition constant determined above, and Bmax of 440 fmol [1251]Leu3a/mg cell protein, representing 130 000 a
-
molecules of [ 25I]Leu3a bound per cell at saturation. Comparison of this value with the number of [1251]Fab' fragments bound per cell (above), as well as the higher affinity of [125I]Leu3a suggest that most of the binding of the intact antibody is bivalent. Due to the higher affinity, efficient removal of cell surface bound [1251]Leu3a required treatment with pH 2 medium or repeated washing at pH 3 (data not shown). Internalization and recycling of radioiodinated Fab' fragments on CD4-transfected HeLa cells By preparing [1251]Fab' fragments of Leu3a, we have developed a monovalent radiolabelled ligand, which binds specifically to CD4 and which can be efficiently removed from the cell surface. This reagent could be used to trace uncrosslinked CD4 on CD4-positive cells and determine the extent of CD4 endocytosis. HeLa-CD4.2G3 cells were labelled on ice (conditions in which endocytosis is inhibited) with sub-saturating concentrations of [1251]Fab' fragments of Leu3a (0.5 nM), washed and then incubated in warmed binding medium at 37°C. At various times, cells were cooled, harvested directly and the total cell-associated [1251]Fab' measured by 'y-counting. Alternatively [1251]Fab' fragments remaining on the cell surface were removed by treatment with low pH medium prior to harvesting and 'ycounting. The result of a typical experiment is shown in Figure 4. The total cell-associated [ 25I]Fab' radioactivity declined over 2 h as a result of dissociation of the bound [1251]Fab' fragments, since the [1251]Fab' was recovered in the medium and >90% could be precipitated with trichloroacetic acid (TCA) even after 2 h at 37°C (not shown). The background level of acid-resistant radioactivity on cells kept on ice represented 8% of the total cell-associated
3643
A.Pelchen-Matthews, J.E.Armes and M.Marsh
Table H. Internalization of CD4 on HeLa-CD4 and NIH3T3-CD4 cells
Acid-resistant (%) t = 60 min t6m-tO
Cell line
Clone
Ligand
Endocytosis assays HeLa-CD4 HeLa-CD4 NIH3T3-CD4 NIH3T3-CD4
2G3 2G3 2D12 2D5
[125I]Fab' ['25I]Leu3a
[125I]Fab' [125j]Fab'
7.5 3.5a 9.3 + 2.4a 3.2b 4.7 6.3
Recycling assays HeLa-CD4 HeLa-CD4 NIH3T3-CD4
2G3 2G3 2D12
[125I]Fab' [125I]Leu3a [125I]Fab'
1. Oa 10.4 7.0 f0.4b 9.6
t = 0
39.9 ± 6. la 49.2 + 4.2a 32.4 3.3b 38.0
32.5 ± 6.6a 1 39.9 l.9a 25.9 4.5b 31.7
9.4a
42.9 + 8.8a 62.5 i0.8b 57.2
53.3 69.5 62.8
0.4b
Cells were labelled for 2 h on ice with [125I]Fab' fragments of Leu3a or with [125I]Leu3a and washed. The amount of acid-resistant label (not removed by washing with BM at pH 3) after this labelling period is shown as the percentage of the total cell-associated activity (t = 0). Subsequently, cells were warmed to 37°C for 60 min (endocytosis assays), or for 30 min, surface stripped and rewarmed for 60 min (recycling assays), before acid-resistant counts were again measured. The difference between the acid-resistant activity after 60 min and that initially at t = 0 represents the minimum amount of internalized ligand. aMean and standard deviation from three experiments. bMean and standard deviation from two experiments.
activity. On warming, the acid-resistant activity increased slowly, suggesting that the bound [1251]Fab' fragments, and hence their CD4 receptor molecules, were removed from the cell surface into an acid-resistant and presumably intracellular compartment. The rate of internalization rep)resented uptake of 1.5-2% of the total cell-associated [1 51]Fab' per minute. When acid-resistant counts were plotted as a proportion of the total cell-associated activity (Figure 4, inset), it was clear that between 30 and 60 min a plateau was reached, with -40% of the cell-associated [1 5I]Fab' resistant to acid stripping. Subtraction of the background acid-resistant activity at t = 0 suggests that at least 30% of the [1251]Fab' was in an internal compartment after 1 h (Table II). The observed plateau in the proportion of internalized [125I]Fab' could indicate a limited capacity of CD4 to internalize or, alternatively, that the internalized ['251]Fab'-labelled CD4 was recycled. To assess directly whether the internalized [1251]Fab' was recycled, labelled cells were allowed to internalize [125I]Fab'-labelled CD4 for 30 min at 37°C. Cell surface [125I]Fab' was then removed with low pH medium before the cells were returned to 37°C. At various times of reincubation, cells were cooled and the total cell-associated or internal [1251]Fab' levels measured as above. As shown in Figure 5, there was a decrease in the total cell-associated radioactivity with reincubation time, the dissociating [1251]Fab' activity being recovered in the medium. Since the cells were washed and surface-stripped prior to reincubation, this material must have come from inside the cells; the recovered radioactivity was still > 80% TCA-precipitable, indicating that there was no significant degradation. In addition, during the reincubation at 37C, a proportion of the total cell-associated [1251]Fab' label again became sensitive to low pH treatment, indicating that bound [125I]Fab' fragments had reappeared on the cell surface. These observations show that the bound [1251]Fab', and hence CD4, is indeed recycled. After 30-60 min a steady state of internalization and recycling was reached, with the internal counts representing -40% of the total cellassociated radioactivity (Table II). -
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