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The Journal of Immunology, 1998, 161: 3551-3556.
Copyright © 1998 by The American Association of Immunologists

lck-Independent Inhibition of T Cell Antigen Response by the HIV gp1201

Sophie Gratton2,*,{dagger}, Michael Julius{ddagger} and Rafick-Pierre Sékaly3,*,§

* Laboratoire d’Immunologie, Institut de Recherches Cliniques de Montréal, Montréal, Québec, Canada; {dagger} Division of Experimental Medicine, Department of Medicine, McGill University, Montréal, Québec, Canada; {ddagger} Department of Immunology, University of Toronto and the Wellesley Hospital Research Institute, Toronto, Ontario, Canada; and § Département de Microbiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a murine T cell clone transfected with either wild-type human CD4 or mutated forms of CD4 to characterize the pathways involved in this inhibitory effect of gp120. Ag-induced proliferation of T cell clones transfected with human CD4 was completely inhibited in the presence of gp120, even though stimulation of this clone is independent of a CD4/MHC class II interaction. In addition, our results demonstrate that the inhibition by gp120 is not due to the sequestration of lck from TCR and does not require activation of lck by gp120. This suggests that CD4 can regulate the initiation of T cell activation independently of its interaction with lck. Moreover, we demonstrate that the nonresponsiveness induced by gp120 can be reversed by soluble CD4 when added early after onset of stimulation and that gp120 exerts its inhibitory effect when cells are in the G0 >= 1 phase of the cell cycle.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4-positive T lymphocytes recognize Ag presented by MHC class II molecules (reviewed in 1 . The interaction between CD4 and MHC class II molecules was first reported to enhance the response of T cells induced by antigenic stimulation. This enhancement has been shown to generally require the association of the cytoplasmic domain of CD4 with the src-related tyrosine kinase p56lck (2, 3, 4). Two cysteine residues at positions 420 and 422 of CD4 mediate its interaction with the N-terminal portion of lck (5, 6, 7). Aggregation of CD4 molecules using CD4-specific Ab leads to an increase in tyrosine kinase activity of the CD4-associated lck (8, 9). The expression of lck is required to initiate T cell activation (10, 11). Indeed, the activity of lck is implicated in the phosphorylation of the {zeta}-chain of the CD3 complex and the subsequent recruitment and activation of the ZAP-70 tyrosine kinase (11, 12, 13). We have previously shown that CD4 can sequester lck and thus inhibit anti-TCR-induced proliferation (14). Conversely, coaggregation of the CD4/lck complex with the TCR through the simultaneous interaction with the same MHC class II molecule is required to trigger optimal T cell activation (14, 15).

Signals generated through the CD4/lck complex can also negatively regulate T cell activation. Indeed, aggregation of CD4 with Ab, independently of the TCR/CD3 complex, inhibits T cell activation, leading to a state of anergy and/or to priming of T cells for apoptosis (16, 17, 18, 19). Since the CD4 molecule is the physiologic receptor for HIV, this negative signal could also contribute to the pathogenicity of HIV infection. Indeed, gp120 was demonstrated to induce lck activity, tyrosine phosphorylation of cellular substrates, calcium influx, and the expression of activation markers such as HLA-DR and the IL-2 receptor {alpha}-chain (20, 21, 22, 23). However, treatment of CD4-positive human T cells with gp120 inhibits stimulation induced by mAb specific for TCR or specific Ag, leading to the induction of anergy (24, 25, 26, 27, 28).

Previous reports have also shown that the enhancement of CD4-dependent T cell activation does not always require its association with lck, suggesting a regulatory role for the extracellular domain of CD4 (29, 30, 31). In this context, one cannot rule out the possibility that gp120 could interfere with the function of CD4-positive T cells through an lck-independent mechanism. We have used a T cell clone that does not require the association of CD4 with lck for its Ag-specific response to monitor the role of this association in gp120 inhibition of T cell activation. Our results indicate that the association of CD4 with lck is not required for the inhibitory effect of gp120, further confirming the critical role of the extracellular domain of CD4 in regulating T cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

Wild-type CD4 and CD4 mutants were generated by PCR (32) and cloned into the MNCstuffer retroviral expression vector coding for the neomycin resistance gene (a gift from Brian Seed, Massachusetts General Hospital, Boston, MA). Transfection of the packaging cell line DAMP was performed to produce the proper infectious supernatants. 2.10 cells were then infected, and stable transfectants were selected for in G418 (1 mg/ml). 2.10 cells were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with IL-2 and lecithin as previously described (14). C57BL/6 mice (4–6 wk old) were purchased from Charles River (Quebec, Canada). Splenocytes were isolated from the mice as previously described (14). Recombinant gp120 BH10 (gp120) was obtained from A. Truneh of Smith Kline Beecham (King of Prussia, PA) while recombinant soluble CD4 (rCD4) was obtained from Genentech (San Francisco, CA).

Antigenic stimulation

T cells (5 x 104) were stimulated with 5 x 105 C57BL/6 syngeneic irradiated splenocytes (2500 rad) and OVA (Sigma, St. Louis, MO) at the indicated concentrations. Recombinant gp120 or soluble CD4 were added when mentioned at a final concentration of 5 µg/ml. When mentioned in the text, epidermal growth factor (Upstate Biotechnology, Lake Placid, NY) was added at a final concentration of 100 nM. Cells were stimulated for 40 h at 37°C and then pulsed for 6 h with 1 µCi of [3H]thymidine (Mandel Scientific, Ontario, Canada). Thymidine uptake was quantified by liquid scintillation spectroscopy using the ß-plate counter (Pharmacia, Quebec, Canada).

Synchronization and cell cycle analysis

Cells were synchronized by starving them of IL-2 for 2 h at 37°C and further culturing them in limiting amounts of IL-2 (0.05%) for 12 h at 37°C. For cell cycle analysis, cells were stained using a modified Krishan buffer. Briefly, cells were fixed in 50% ethanol. Cells were then incubated in modified Krishan buffer (0.1% sodium citrate, 0.02 mg/ml RNase, 0.3% Nonidet P-40, 0.05 mg/ml propidium iodide) for 30 min on ice. Cells were then centrifuged and resuspended in fresh Krishan buffer. Samples were analyzed on a FACStarPlus (Becton Dickinson, Mountain View, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
gp120 inhibits antigenic stimulation of a murine T cell clone transfected with human CD4

A CD4-negative murine T cell clone, 2.10, which recognizes OVA presented by I-Ab, was transfected with human CD4 to characterize the mechanism of inhibition of antigenic stimulation by gp120. This T cell clone was previously shown to be CD4 independent since it proliferates in response to stimulation by Ag to the same extent whether it expresses CD4 or not (Ref. 14 and Fig. 1GoA). As shown in Figure 1Go, gp120 inhibited over 95% of Ag-induced proliferation of a human CD4-positive 2.10 clone whereas it did not affect the response to Ag of the CD4-negative 2.10. Furthermore, inhibition was observed even at the highest concentrations of Ag tested (50 to 500 µg/ml), demonstrating the high potency of gp120 to inhibit antigenic stimulation (Fig. 1GoB).



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  		FIGURE 1. A, gp120 inhibits antigenic stimulation. T cells (5 x 104), either CD4-positive (CD4) or -negative (2.10) were incubated with 5 x 105 irradiated C57BL/6 splenocytes and 200 µg/ml of OVA, in the presence or absence of gp120, for 40 h at 37°C. Cells were then pulsed with 1 µCi of [3H]thymidine for 6 h before harvesting. B, gp120 inhibits activation by a wide range of Ag concentrations. CD4-positive T cells (5 x 104) were incubated with 5 x 105 irradiated C57BL/6 splenocytes and OVA at the indicated concentrations, in the presence (gp120) or absence (NS) of 5 µg/ml gp120, for 40 h at 37°C. Cells were then pulsed with 1 µCi of [3H]thymidine for 6 h before harvesting.

 
An increase in lck activity is not sufficient to inhibit Ag-induced proliferation

gp120 was previously demonstrated to increase the tyrosine kinase activity of CD4-associated lck (20, 21, 22, 23, 28). To evaluate the potential contribution of lck activity in inhibition by gp120, we have transfected the 2.10 clone with a chimeric molecule consisting of the extracellular domain of the epidermal growth factor (EGF)4 receptor and the transmembrane and cytoplasmic domains of human CD4. We have previously demonstrated that this chimera is associated with a similar amount of lck as wild-type CD4.5 Moreover, binding of EGF to the chimera activates lck tyrosine kinase activity.5 Two independently derived clones expressing the chimera were tested several times in functional assays for their capacity to respond to Ag. Results of a representative experiment are shown in Figure 2Go. These clones responded to Ag as efficiently as cells expressing wild-type CD4 molecules, further confirming the lack of requirement of a CD4-MHC class II interaction. Interestingly, addition of saturating levels of EGF (100 nM), which induces the tyrosine kinase activity of lck, does not have any effect on the response to OVA. Based on these results, it was tempting to speculate that the inhibition by gp120 of T cell activation did not require activation of lck associated with the cytoplasmic tail of CD4.



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  		FIGURE 2. EGF does not inhibit stimulation of chimera-expressing clones. Either EC1 or EC4 T cells (5 x 105), which express the EGFRCD4 chimera, were stimulated with 100 µg/ml OVA presented by 5 x 105 irradiated C57BL/6 splenocytes, in the presence of EGF (UBI) at 100 nM or media (NS), for 40 h at 37°C. Cells were then pulsed with 1 µCi of [3H]thymidine for 6 h before harvesting.

 
The inhibition of antigenic stimulation by gp120 does not require the association of lck with CD4

To verify if the association of CD4 with lck is required for the inhibition of gp120, several 2.10 clones expressing a mutant CD4 molecule that is not associated with lck, C4202A, were stimulated in the presence or in the absence of gp120. Each clone was tested at least twice, and a representative experiment is shown in Figure 3GoB. Interestingly, gp120 inhibited proliferation of C4202A clones induced by stimulation with OVA. This result clearly demonstrates that the inhibition of antigenic stimulation induced by gp120 does not require the association of lck with CD4.



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  		FIGURE 3. A, Cell surface expression of CD4 transfectants. Cells were labeled with CD4-specific mAb 1F3 followed by an FITC-coupled GAM. Dotted lines represent the negative control for each transfectant. B, gp120 inhibits stimulation of C4202A and CD4 3S clones. T cells (5 x 104) were incubated with 5 x 105 irradiated C57BL/6 splenocytes and 200 µg/ml of OVA, in the presence (gp120) or absence (NS) of gp120 at 5 µg/ml, for 40 h at 37°C. Cells were then pulsed with 1 µCi of [3H]thymidine for 6 h before harvesting. The differences in thymidine incorporation between transfectants stimulated in the absence of gp120 are considered nonsignificant and due to clonal variation (data not shown and Ref. 14).

 
Binding of gp120 to CD4 has been reported to induce the internalization of CD4, leading to the suggestion that this internalization plays a role in the inhibitory effect of gp120 (24). Three serine residues located in the cytoplasmic domain of CD4 are responsible for the internalization and degradation of CD4 molecules (33). These serine residues were mutated to alanine residues to abrogate the internalization of CD4 (32). Several clones expressing this mutant were derived. Interestingly, these clones still responded to Ag stimulation (Fig. 3GoB) whereas this mutation was previously found not to restore a CD4-dependent response (4), further confirming the CD4-independent nature of this clone. Furthermore, gp120 inhibited Ag-induced proliferation of these transfectants (Fig. 3GoB). This result suggests that inhibition of antigenic stimulation by gp120 does not require the triple serine endocytosis motif of the cytoplasmic tail of CD4, providing further evidence for the lack of role of the cytoplasmic domain of CD4 in this function.

gp120 prevents Ag-mediated blastogenesis

To further determine the mechanism leading to gp120-mediated inhibition of T cell activation, we verified whether gp120 was capable of preventing early activation signals, such as blast transformation. Clones expressing wild-type CD4 were stimulated with Ag in the presence or absence of gp120 for 24 h, and their size was monitored using the FACScan. As a control, an aliquot of cells was incubated without Ag or IL-2 for the same period. As depicted in Figure 4Go, A and B, cells exposed to Ag in the absence of gp120 had an increased FSC as compared with cells stimulated with Ag in the presence of gp120, indicating that the presence of gp120 prevented blast transformation in response to antigenic stimulation. Further analysis was conducted using FSC/SSC contour plots of total cells (Fig. 4Go, A–C) or with dead cells gated out using propidium iodide (Fig. 4Go, D–F). The lower left quadrant of each plot contains the splenocytes used to present Ag while blasts accumulate in the upper right quadrant. Fig. 4Go, A and D, indicates that cells stimulated with Ag undergo blast transformation and exclude propidium iodide. On the other hand, cells stimulated by Ag in the presence of gp120 did not increase in size (Fig. 4GoB). Figure 4GoE indicates that cells die in the presence of gp120, since they are totally excluded by propidium iodide staining. This is further confirmed by the fact that the profiles are similar to those observed with cells that were incubated without any Ag or IL-2 (Fig. 4Go, C and F). This T cell clone is dependent on exogenous IL-2 for growth and, upon deprivation of IL-2, cells undergo apoptosis (data not shown). Hence, cell death observed in the presence of gp120 may be due either to a direct effect of gp120 on T cells or to apoptosis resulting from the lack of stimulation and the absence of autocrine IL-2.



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  		FIGURE 4. gp120 prevents blasting of cells and leads to cell death. Cells were stimulated with OVA at 200 µg/ml (A, B, D, and E) or media (C and F) in the presence of gp120 at 5 µg/ml (B and E) or media (A, D, C, and F) and irradiated C57BL/6 splenocytes for 24 h. Cells were harvested and analyzed using the FACscan. A–C, Show the FSC/SSC profiles of total cells. D–F, Show the FSC/SSC profiles of populations in which dead cells are gated out using propidium iodide.

 
gp120 inhibition is reversible by soluble CD4

To determine whether cell death observed in the presence of gp120 was due directly to the binding of gp120 to CD4, we verified whether soluble CD4 could reverse the inhibition induced by gp120. As shown in Figure 5GoA, soluble CD4 completely prevented inhibition of antigenic stimulation by gp120 when added at the onset of stimulation. Interestingly, soluble CD4 completely restored Ag-induced proliferation when added 6 h after start of stimulation, while partially restoring the response when added 18 or 24 h after addition of Ag and APC. This partial reversal of inhibition cannot be due to lack of Ag, since addition of fresh splenocytes and Ag with the soluble CD4 at 24 h still does not completely restore the response (data not shown).



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  		FIGURE 5. A, Soluble CD4 reverses the inhibition of stimulation induced by gp120. Cells expressing wild-type CD4 were stimulated with OVA at 200 µg/ml presented by C57BL/6 irradiated splenocytes in the presence (gp120) or absence (NS) of gp120 at 5 µg/ml. Soluble CD4 at 5 µg/ml was added at 0, 6, 18, or 24 h poststimulation as indicated in the legend. Total stimulation time was 40 h before [3H]thymidine incorporation. B, Time course of addition of gp120. Cells expressing different CD4 constructs were stimulated with OVA at 100 µg/ml. gp120 was added at 5 µg/ml at the onset of stimulation or 24 h later. [3H]Thymidine incorporation was performed 40 h after initiation of stimulation. The differences in thymidine incorporation between transfectants stimulated in the absence of gp120 are considered nonsignificant and due to clonal variation (data not shown and Ref. 14).

 
These results argue against a direct cytopathic effect of gp120 on T cells but rather support the hypothesis that gp120 prevents the initiation of T cell activation. The observed cell death would thus be due to a consequent lack of IL-2, and ensuing apoptosis. Moreover, the state of nonresponsiveness induced by gp120 is not permanent, since it can be reversed by the addition of soluble CD4.

The presence of gp120 is required at the G0/G1 phase of the cell cycle for efficient inhibition of T cell activation

In light of the above results demonstrating only partial rescue of gp120-mediated inhibition by soluble CD4 when added 24 h after the onset of stimulation, we were interested in determining the time frame in which gp120 had to be present during stimulation to inhibit T cell activation. It was also interesting to verify if cells expressing the different CD4 mutants exhibited the same requirements. As demonstrated in Figure 5GoB, gp120 inhibited the response of clones expressing wild-type or mutant forms of CD4 (C4202A and CD4 3S) when added at the onset of stimulation. Remarkably, when gp120 was added at 24 h poststimulation, we could still observe a significant inhibition of antigenic stimulation. As shown in Figure 6GoA, at the beginning of stimulation, cells are distributed in all phases of the cell cycle. We thus hypothesized that cells that are in the S, G2, and M phases at the onset of stimulation must thus complete their cycle back to G0/G1 before encountering Ag and becoming susceptible to inhibition by gp120.



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  		FIGURE 6. A and B, Cell cycle analysis of unsynchronized (A) and synchronized (B) wild-type CD4-expressing cells. Cells were stained using Krishan buffer as described in Materials and Methods and analyzed using the FACStarPlus (Becton Dickinson). C, Time course of addition of gp120. Synchronized and unsynchronized cells were stimulated with OVA at 200 µg/ml only (NS) or with addition of gp120 at 5 µg/ml at 0, 9, or 24 h poststimulation. Total stimulation length is 40 h before a 6-h pulse with [3H]thymidine.

 
To verify this hypothesis, we enriched for G0/G1 CD4-positive cells and compared their susceptibility to inhibition by gp120 with that of unsynchronized population. To synchronize cells, we first starved them of IL-2 for 2 h and then let them grow for 12 to 16 h in limiting amounts of IL-2 (5% of concentration used for expansion) before stimulation. As shown in Figure 6Go, A and B, this treatment led to an accumulation of cells in the G0/G1 phase of the cell cycle (75–80%), as opposed to unsynchronized cells that were distributed more evenly in all phases of the cycle (40–45% in G0/G1). Both populations were stimulated with Ag, and gp120 was added at different time points after the onset of stimulation (Fig. 6GoC). While gp120 profoundly inhibited stimulation of both populations when added at initiation of stimulation, addition of gp120 9 h later inhibited only 56% of the response of the "synchronized" population, whereas it inhibited 95% of the response of the unsynchronized population. Addition of gp120 at 24 h poststimulation further differentiated the two populations; while we could observe a 75% inhibition in the unsynchronized population, the "synchronized" population was only weakly inhibited (13%). These results suggest that all "synchronized" cells have seen Ag and become committed for activation before the 24-h time point and thus cannot be inhibited by addition of gp120 at that time point. On the contrary, in the unsynchronized population, some cells are not stimulated yet at the 24-h time point. These cells are the ones that were in the S/G2/M at time 0 and are now in G0/G1 and susceptible to inhibition by gp120 24 h after start of stimulation. Analysis of the populations 24 h after onset of stimulation could not allow a distinction between cells committed to G1 and cycle progression and those not stimulated and residing in G0 (data not shown). These results support the hypothesis that gp120 must be present in the G0/G1 phase of the cycle to inhibit antigenic stimulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have extended the characterization of the effect of gp120 on T cell activation by demonstrating that gp120 can also abrogate antigenic stimulation of a murine T cell clone transfected with human CD4. The concentrations of gp120 required to observe this inhibition (5 µg/ml) are in the range of levels found in HIV-infected patients (34). Whether the monomeric form or an oligomeric form of gp120 is required to observe inhibition of activation is still controversial. In our hands, the monomeric form of gp120 was sufficient to completely inhibit Ag-induced stimulation, confirming that aggregation of CD4 is not required for this effect. A decrease in T cell stimulation by mitogens or recall Ags is observed in HIV-infected patients (35, 36). The HIV env glycoprotein gp120 has been suggested to play a role in this phenomenon of the inhibition of T cell activation. Indeed, soluble or membrane-bound forms of gp120 can inhibit in vitro TCR-triggered activation of both human primary PBL and T cell lines. This inhibition could result in vivo from the interaction of gp120 at the surface of infected cells or of gp120 on virions with CD4 molecules. The presence of viral particles in lymph nodes where T cells are activated by their encounter with specific Ag supports this model.

The inhibitory effect of gp120 was completely reversible in the presence of soluble CD4 when added early after triggering of T cell activation. This result shows that, to inhibit stimulation by Ag, a prolonged interaction between CD4 and gp120 is required. However, synchronization experiments and kinetics of addition of either gp120 or soluble CD4 suggest that gp120 exerts its inhibitory effect when cells are in the G0/G1 phase of the cycle, i.e., when TCR recognition of Ag occurs (reviewed in 37 . Indeed, when cells have become committed to cycle at time t = 24 h poststimulation, gp120 was no longer able to abrogate the proliferation of activated T cells. Several cell cycle-regulating genes, such as the cyclins and their kinases, have been implicated in cell cycle progression (reviewed in 38 . Modulation of their activity occurs both at transcriptional and posttranscriptional levels and involves phosphorylation events. Blocking of these kinases by chemicals such as rapamycin and FK506 results in abrogation of cell cycle progression leading to T cell anergy (reviewed in 39 . Interestingly, signals generated through CD4 have been implicated in the regulation of early T cell activation events, and gp120 could thus disrupt one or more of these signals to abrogate T cell stimulation by Ag (40, 41).

The response of this T cell clone to antigenic stimulation is not enhanced by the presence of CD4 (14). The inhibitory effect of gp120 cannot thus be explained by the disruption of an adhesion interaction between CD4 and MHC class II molecules. Binding of gp120 to CD4 has been previously reported to induce intracellular biochemical events such as an increase in tyrosine kinase activity of lck and down-regulation of cell surface expression of CD4. However, our results clearly show that, although inhibition by gp120 is accompanied by an increase in lck tyrosine activity (20, 21, 22, 23, 28), neither its association with CD4 nor an increase in its tyrosine kinase activity are required for the abrogation of Ag-induced T cell proliferation. Indeed, stimulation of cells expressing either the mutant that has lost the association with lck or the endocytosis mutant was inhibited by gp120, whereas activation of lck through the EGFRCD4 chimera had no effect on T cell stimulation. We have previously shown that CD4 can sequester lck and inhibit anti-TCR-induced proliferation if not coaggregated with the TCR (14). The mechanism of inhibition by gp120 was also proposed to involve lck sequestration in T cell lines (42). The inhibition of Ag-induced stimulation by gp120 that we here observe cannot be explained by such a sequestration of lck since clones expressing a mutant of CD4 that does not associate with lck are also inhibited by gp120. Interestingly, treatment with CD4-specific Ab also inhibits antigenic stimulation of T cells expressing a mutant of CD4 that is not associated with lck (29). Taken together, the results presented here define a functional role of CD4 that is independent of its association with lck and its interaction with MHC class II molecules, although mapping to its external portion. Binding of gp120 to the external domain of CD4 could mask this regulatory domain and prevent T cell activation.

It was recently reported that gp120 binds to monomeric forms of CD4 whereas MHC class II molecules induce the dimerization of CD4 molecules through their external domain (43). A possible mechanism of inhibition of gp120 could be the disruption of CD4 dimers, thus modulating CD4 signaling capacity. Alternatively, gp120 could modulate the function of another T cell surface molecule which is associated with CD4 and implicated in early T cell activation events. Indeed, CD4 molecules have been found associated with the tyrosine phosphatase CD45 and the TCR complex (30, 31, 44, 45, 46, 47, 48). It is thus conceivable that binding of gp120 to CD4 could prevent an extracellular interaction between CD4 and the TCR necessary to stabilize the interaction between the TCR and MHC class II molecules (30, 31). Alternatively, gp120 binding to CD4 could sterically prevent an Ag-specific TCR/MHC class II interaction or disrupt a regulatory interaction between CD4 and CD45. This last scenario would lead to modulation of CD45 activity and inhibition of T cell activation (49, 50). Modulation of T cell activation through the CD4-CD45 interaction has been previously reported (49, 50, 51, 52).

Overall, we have demonstrated that inhibition of antigenic stimulation by gp120 is not mediated through direct activation of the CD4-associated lck and occurs in the absence of lck association with CD4. We propose that gp120 modulates in a reversible manner, a regulatory function of CD4 that occurs in the G0/G1 phase of the cell cycle and that is mapped to its extracellular domain.


   Acknowledgments
 
We acknowledge Claude Cantin and Martine Dupuis for cell sorting and cell cycle analysis, and Jacques Thibodeau for critical reading of the manuscript.


   Footnotes
 
1 S.G. was supported by a Medical Research Council (MRC) studentship. R.-P.S. holds an MRC scientist award. This work was supported by grants from the MRC/Industry (UI-12373) and the National Health Research and Development Program (6605–4327-AIDS). Back

2 Current address: Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue du Dr Roux, Paris 75015, France. Back

3 Address correspondence and reprint requests to Dr. Rafick-Pierre Sékaly, Laboratoire d’Immunologie, Institut de Recherches Cliniques de Montréal, 110 ave des Pins ouest, Montréal, Québec, Canada, H2W1R7. E-mail address: Back

4 Abbreviations used in this paper: EGF, epidermal growth factor; FSC, forward light scatter; SSC, side light scatter. Back

5 S. Gratton, L. Haughn, R.-P. Sékaly, and M. Julius. The extracellular domain of CD4 regulates the initiation of T cell activation. Submitted for publication. Back

Received for publication July 21, 1997. Accepted for publication May 27, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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