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Research Center in Infectious Diseases, Laval University Hospital Research Center, and Faculty of Medicine, Laval University, Quebec
Robarts Research Institute and the Departments of Microbiology and Immunology, and Medicine, the University of Western Ontario, London, Ontario, Canada
CD28 is constitutively expressed on CD4+ cells, but its homologue CD152 is only weakly expressed after cell activation. To determine whether these 2 costimulatory molecules can be inserted into human immunodeficiency virus type 1 (HIV-1), virus was produced in CD28- and CD152-expressing Jurkat-derived cells. Both molecules were efficiently acquired by virions. Virus attachment and infectivity were more affected by CD152 than by CD28. Given that CD28/CD152-CD80/CD86 interactions play a dominant role in antigen presentation, it can thus be proposed that the association between virus-anchored host CD28/CD152 and cell-surface CD80/CD86 on target cells might have consequences for the transmission and pathogenesis of HIV-1.
It is now well established that HIV-1 acquires a vast collection of cellular membrane proteins during budding [1]. Although the precise impact of such nonviral constituents on the pathogenesis of HIV-1 remains to be clearly defined, numerous in vitro studies have convincingly shown that the insertion of such host-encoded molecules within mature HIV-1 particles affects the virus replication cycle in several ways.
CD28 is expressed on the surface of T cells as a homodimer of 44 kDa. It has recently been demonstrated that attachment and entry processes were both promoted after the acquisition of CD28 by HIV-1 when target cells express the 2 physiological ligands of CD28, CD80 (B7.1) and CD86 (B7.2) [2]. CD152 (CTLA-4), as a homologue of CD28, presents structural similarity and binds to the same ligands. However, the roles played by these 2 T cellspecific surface molecules is fundamentally distinct [3]. Previous studies have illustrated that CD28 provides the cosignal required for the full activation of T cells on the engagement of the T cell receptor, whereas CD152 acts as an attenuator of T cell activation. Moreover, their level of expression on the surface of CD4+ cells, 1 of the 2 major sources of HIV-1 in infected individuals, differs greatly. Indeed, CD28 is constitutively expressed on the surface of virtually all CD4+ cells, whereas CD152 is induced after cell activation, and its surface expression is limited by a constant reinternalization from the plasma membrane by the adaptor molecule AP-2 [4].
In the present study, we investigated the possible effect of HIV-1 infection on the cell-surface expression of CD28 and CD152, as well as the efficiency of incorporation of both molecules in HIV-1. We present evidence that acute virus infection of an established human T cell line results in a down-regulation of CD28 and a slight up-regulation of cell-surface CD152 expression. Interestingly, both cell-surface constituents are acquired by HIV-1, a process that positively affects virus attachment and production.
Materials and methods.
Jurkat T cells, which naturally express CD28 but not endogenous CTLA-4, were stably transfected with the CTLA-4encoding vector pBIG2i, giving rise to the Jurkat CD28+/CD152+ cell line. This plasmid contains a hybrid bidirectional tetracycline-responsive promoter element that directs the expression of both CTLA-4 and the rtTAN tetracycline-responsive transactivator. RAJI is an Epstein-Barr viruscarrying B cell line that expresses high levels of surface CD80 and CD86 but is negative for CD4, CD28, and CD152 (data not shown). The LuSIV cell line is stably transfected with the SIVmac239 long-terminal repeat (nt -225 +149), which is cloned upstream of the firefly luciferase reporter gene (supplied by J. E. Clements, Johns Hopkins University School of Medicine, Baltimore, MD). Flow-cytometric analyses revealed that RAJI and LuSIV cells express high levels of both CD80 and CD86 molecules (data not shown).
The monoclonal antibody (MAb) 9.3 is specific for human CD28 and inhibits the interaction between CD28 and CD80/CD86 (provided by J. A. Ledbetter, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ). The CTLA-4specific MAbs 11D4 and 20A were obtained from R. Peach (Bristol-Myers Squibb Pharmaceutical Research Institute) and Pharmingen, respectively. The MAb L307.4 is specific for CD80 (Pharmingen), whereas BU-63 is directed against CD86 (supplied by D. L. Hardie, The University of Birmingham, Birmingham, UK). The expression of CD28 and CD152 on the surface of uninfected and virus-infected Jurkat cells was monitored by use of anti-CD28 (9.3) and anti-CD152 (20A) MAbs before cytofluorometric analyses (EPICS Elite ESP; Coulter Electronics).
NL4-3 is an infectious molecular clone that codes for a prototypic X4-tropic variant of HIV-1. Fully competent NL4-3 particles were produced by calcium phosphate transfection in human 293T cells, as described elsewhere [2]. Preparations were normalized for virion content by use of an in-house double-antibody sandwich ELISA specific for the major viral p24 protein. NL4-3 was produced in Jurkat cells by first incubating such cells with HIV-1 (100 ng of p24/1 × 106 cells) for 3 h at 37°C. Cells were then washed twice with PBS and resuspended in RPMI 1640 (1 × 106 cells/mL) with or without doxycyclin (100 ng/mL). On days 2, 4, and 6 after infection, cells were centrifuged, and virus-containing supernatants were filtered to remove cellular debris, aliquoted, and stored at -85°C. The presence of host-encoded CD28 and CD152 in HIV-1 was monitored by use of streptavidin-coated magnetic beads in combination with biotinylated anti-CD28 (clone 9.3) or anti-CD152 (clone 11D4) antibodies, as described elsewhere [2].
Virus stocks (5 ng of p24) were exposed to RAJI cells (2.5 × 105) for 1 h at 37°C in a total volume of 200 L of complete culture medium in a 96-well tissue-culture plate. Next, cells were washed 3 times with 200 L of PBS, and the pellet was resuspended in 200 L of PBS-Tween supplemented with 1% bovine serum albumin before the measurement of the p24 content. In this set of experiments, virus stocks were pretreated with anti-CD28 (9.3) or anti-CD152 (11D4) MAbs (final concentration, 1 g/mL) for 30 min at 37°C or were given no pretreatment. In some experiments, RAJI cells were pretreated with anti-CD80 (L307.4) or anti-CD86 (10A8) antibodies (final concentration, 1 g/mL) for 30 min at 4°C or were given no pretreatment before inoculation with virus preparations.
Virus preparations were pretreated with anti-CD28 (9.3) or anti-CD152 (11D4) MAb for 30 min at 37°C or were given no pretreatment before they were used to infect LuSIV (100 ng of p24 and 1 × 105 cells). Cells were incubated for 24 h at 37°C before they were lysed and tested for luciferase activity by use of a microplate luminometer (MLX; Dynex Technologies). Luciferase activity is expressed as relative light units.
Results.
We used a Jurkat derivative that constitutively expresses CD28 but can also express CD152 in an inducible manner after treatment with doxycyclin [5]. A decreased expression of CD28 coupled with an up-regulation of CD152 expression was detected after HIV-1 infection (figure 1). These findings are in agreement with observations made in clinical studies that have reported a diminished number of CD28-expressing T cells coupled with an up-regulation of CD152 expression on CD4+ cells [6, 7]. Interestingly, the maximum HIV-1mediated effect on CD28 and CD152 was reached 96 h after virus infection, which coincided with the peak of HIV-1 production (i.e., 21 and 455 ng/mL of p24 at 48 and 96 h after infection, respectively).
To scrutinize the possible acquisition of CD152 by nascent HIV-1 particles, virions were harvested from Jurkat CD28+/CD152+ cells on days 2, 4, and 6 after infection and were then subjected to a virus precipitation test. Viruses were efficiently captured in a comparable manner by the 2 antibodies tested (figure 2A). As expected, viruses produced in Jurkat CD28+/CD152- cells (i.e., not treated with doxycyclin) incorporated CD28 but not CD152 (data not shown). We next studied the contribution of these 2 host-derived constituents to virus attachment onto the surface of cells expressing the natural counterreceptors of these 2 molecules (i.e., CD80 and CD86). Ligation of HIV-1 harvested from Jurkat CD28+/CD152+ cells to CD4- RAJI cells was slightly reduced after the addition of an anti-CD28 antibody (figure 2B). Interestingly, virus attachment was almost completely abolished by pretreatment with an anti-CD152 antibody. The importance of interactions between virus-anchored host CD28 and CD152 in the adsorption of HIV-1 particles to the surface of RAJI cells was confirmed when such target cells were pretreated with anti-CD80 and anti-CD86 antibodies. The pretreatment of HIV-1 particles with anti-CD28 antibody had a modest effect on virus infectivity when CD4+ indicator LuSIV cells were used (figure 2C). However, a more significant diminution was seen after the addition of anti-CD152 antibody. Virus attachment and infectivity were similarly affected by anti-CD28 antibody when HIV-1 particles harvested from Jurkat CD28+/CD152- cells were used instead (data not shown).
Discussion.
Because previous studies have revealed that HIV-1 can modulate the surface expression of CD28 and CD152, it is possible that virus-encoded proteins are likely to be directly involved in this process. Among HIV-1 proteins, one that could play a role in the modulation of the cell-surface expression of CD28 and CD152 is Nef. It has been shown that the accessory protein Nef diminishes the cell-surface expression of CD4 and major histocompatibility complex class I molecules through clathrin-dependent endocytosis [8]. More relevant to the present study, Swigut et al. [9] have already suggested the involvement of Nef in the HIV-1mediated decrease of CD28. In the case of CD152, the natural endocytosis pathway uses clathrin-coated pits and requires the presence of the tyrosine residue 165 in its unphosphorylated form, which permits the association of the 2 subunit of the AP-2 complex with the cytoplasmic tail of CD152 [4, 10, 11]. Interestingly enough, CD152 and Nef share a highly conserved sequence of 21 aa in their C-terminal portion [12]. This sequence contains the potential AP-2 binding motif YXXO, which is highly conserved in CD152 (Y165VKM) but not in HIV-1 Nef. Also, in this sequence, both CD152 and Nef bear a putative SH3 binding site (PxxP) that is well conserved. How Nef could interfere in the process of CD152 endocytosis is still unknown, but it can be proposed that Nef and CD152 compete for the endocytic apparatus. It is not known whether Nef has the ability to bind directly to CD152, as is most likely the case for CD28. One possibility is that Nef favors the recruitment of the endocytic complex for CD28 after a direct interaction between Nef and CD28, as shown for CD4. On the other hand, if Nef is unable to bind to CD152, it could limit the availability of the endocytosis machinery to CD152, which would result in an up-regulation of the surface expression of this membrane protein.
It is known that HIV-1 down-regulates some cell-surface proteins that are crucial for the establishment of a potent immune response. As for CD28 and CD152, the tightly controlled regulation of their expression on the surface of T cells is essential for the establishment and control of a proper immune response. Thus, the observation that HIV-1 infection modulates the expression pattern of CD28 and CD152 has led to some speculation about the advantages and/or the consequences of this phenomenon. First, it has been suggested that the HIV-1associated down-regulation of CD28 expression could reduce the adhesion of virus-infected T cells to antigen-presenting cells (APCs) and thereby facilitate the propagation of infection to other APCs [9]. With regard to the results obtained in the present study, this scenario is unlikely, because the down-regulation of CD28 expression on the cell surface correlated with an up-regulation of CD152 expression, a molecule that displays a greater affinity for CD80 and CD86 than does CD28 (see below for more details). Second, we have demonstrated that CD152, although weakly expressed on the cell surface, is efficiently incorporated within HIV-1 particles. The up-regulation of this protein on the cell surface, as well as its possible coordinated cell-surface delivery with virus-encoded envelope proteins at the time of HIV-1 infection [13], could favor its insertion into newly formed virions. On the other hand, the presence of CD28 and CD152 on the surface of HIV-1 could confer to the viral particle the capacity to induce CD80/CD86-mediated signal-transduction pathways in APCs. It is of interest to note that the CD152-mediated ligation of CD80 and/or CD86 on dendritic cells activates tryptophan catabolism, a process that leads to immunosuppression [14]. Studies are needed to define whether CD152-bearing virions can modulate tryptophan catabolism in dendritic cells.
There is, at present, a growing interest in research aimed at deciphering cell-surface receptors implicated in the attachment of mature HIV-1 to the cell surface. Because virus binding is a multistep process of great complexity that can affect HIV-1 pathogenesis by either facilitating new rounds of infection in cis or promoting the infection of permissive cells in trans, a better understanding of the importance of nonviral constituents in the initial step of HIV-1 replication is crucial for the design of new drugs.
Acknowledgments
We thank Dr. M. Dufour, for technical assistance in flow-cytometric studies, S. Méthot, for editorial assistance; and, especially, C. Cté, for technical support. This work was performed by J.-F.G. in partial fulfillment of a Ph.D. degree in the Microbiology-Immunology Program, Faculty of Medicine, Laval University. M.J.T. holds the Canada Research Chair in Human Immuno-Retrovirology (Tier 1 level).
References
1. Tremblay MJ, Fortin JF, Cantin R. The acquisition of host-encoded proteins by nascent HIV-1. Immunol Today 1998; 19:34651. First citation in article
2. Giguère JF, Paquette JS, Bounou S, Cantin R, Tremblay MJ. New insights into the functionality of a virion-anchored host cell membrane protein: CD28 versus HIV type 1. J Immunol 2002; 169:276271. First citation in article
3. Acuto O, Michel F. CD28-mediated co-stimulation: a quantitative support for TCR signalling. Nat Rev Immunol 2003; 3:93951. First citation in article
4. Shiratori T, Miyatake S, Ohno H, et al. Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP-2. Immunity 1997; 6:5839. First citation in article
5. Baroja ML, Luxenberg D, Chau T, et al. The inhibitory function of CTLA-4 does not require its tyrosine phosphorylation. J Immunol 2000; 164:4955. First citation in article
6. Leng Q, Bentwich Z, Magen E, Kalinkovich A, Borkow G. CTLA-4 upregulation during HIV infection: association with anergy and possible target for therapeutic intervention. AIDS 2002; 16:51929. First citation in article
7. Steiner K, Waase I, Rau T, Dietrich M, Fleischer B, Broker BM. Enhanced expression of CTLA-4 (CD152) on CD4+ T cells in HIV infection. Clin Exp Immunol 1999; 115:4517. First citation in article
8. Marsh M, Pelchen-Matthews A. Endocytosis in viral replication. Traffic 2000; 1:52532. First citation in article
9. Swigut T, Shohdy N, Skowronski J. Mechanism for down-regulation of CD28 by Nef. EMBO J 2001; 20:1593604. First citation in article
10. Bradshaw JD, Lu P, Leytze G, et al. Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin-associated protein is negatively regulated by tyrosine phosphorylation. Biochemistry 1997; 36:1597582. First citation in article
11. Chuang E, Alegre ML, Duckett CS, Noel PJ, Vander Heiden MG, Thompson CB. Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression. J Immunol 1997; 159:14451. First citation in article
12. Vieira A. Structural similarity of two T cell signaling regulators suggests a conserved and interactive mechanism of immunosuppression. Mol Immunol 1998; 35:8814. First citation in article
13. Miranda LR, Schaefer BC, Kupfer A, Hu Z, Franzusoff A. Cell surface expression of the HIV-1 envelope glycoproteins is directed from intracellular CTLA-4-containing regulated secretory granules. Proc Natl Acad Sci USA 2002; 99:80316. First citation in article
14. Fallarino F, Grohmann U, Hwang KW, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol 2003; 4:120612. First citation in article