Activation of HIV expression by CD30 triggering in CD4+ T cells from HIV-infected individuals

Immunity,

Vol. 3. 251-255,

August,

1995, Copyright

0 1995 by Cell Press

Activation of HIV Expression by CD30 Triggering in CD4+ T Cells from HIV-Infected Individuals Enrico Maggi,’ Francesco Annunxiato,’ Roberto Manetti,’ Roberta Biagiotti; Maria Graxia Giudizi,’ Adriana Ravina,’ Fabio Almerigogna,t Norman Boiani,t Mark Alderson,* and Sergio Romagnani’ l lstituto di Clinica Medica 3 University of Florence Viale Morgagni, 85 50134 Firenze Italy tlstituto di Clinica Medica 1 University of Pisa Italy Slmmunex Research and Development Corporation 51 University Street Seattle, Washington 98101.2936

Summary CD30 is a member of the tumor necrosis factor receptor superfamily, preferentially expressed by T cells producing type 2 helper (Th2) cytoklnes, whose ligand (CD3OL) has been identlfled on B cells, activated macrophages, and a subset of activated T cells. We show here that cross-linking CD30 with an agonlstic CD30specific monoclonal antibody, as well as with CD30L+ CD8+ T cell clones or CD3OL+ B cells, enhanced HIV replication in CD4+ T cells from HIV-Infected indivlduals, and such a potentiatlng effect was inhibited by anti-CD30L antibody. The anti-CD3OL antibody also exerted a suppressive effect on spontaneous HIV replication occurring in lymph node cells from an HIV-seropositive patient, showing CD30L expression by both B and CD8+ T lymphocytes. Thus, CD30 trlggerfng by CD30L-expressing cells may play an Important role In the activation of HIV expression from latently infected CD4 + T cells. Introduction It has been suggested that during HIV infection there is a bias toward type 2 helper (ThP)-like responses and hence Thl inhibition, which may contribute to the loss of control of the immune system over HIV infection, resulting in progression to AIDS (Clerici et al., 1993). In recent studies, we and others were unable to support the concept of a general massive alteration to a Th2 pattern in HIV-infected individuals (Maggi et al., 1994a; Graziosi et al., 1994). However, large numbers of CD8+ T cell clones showing a clear-cut Th2-like cytokine profile were generated from both peripheral blood and skin biopsies of subjects in advanced phases of HIV infection (Maggi et al., 1994b); moreover, we found that HIV replicates preferentially in Th2 and ThO rather than Thl CD4+ T cell clones in vitro (Maggi et al., 1994a). More recently, we have shown that CD4+ T cell clones producing ThP-type cytokines (i.e., Th2

and, to a lesser extent, ThO cells), but not Thl cells, express CD30 (Del Prete et al., 1995) one of the members of the tumor necrosis factor-nerve growth factor (TNFNGF) receptor superfamily (Smith et al., 1990; Durkop et al., 1992). The ligand for CD30 (CD3OL) has been recently cloned and found to be expressed on activated macrophages and a subset of activated T cells (Smith et al., 1993). In normal conditions there are not CD30+ cells in the blood, whereas they are present at scanty number as large mononuclear cells with evident nucleolus, mainly around the B cell follicles of lymphoid tissues and, to a minor degree, at the edge of germinal centers (Stein et al., 1985). The extracellular portion of CD30 is proteolitically cleaved to produce a soluble form of the molecule (sCD30), which is released by CD3O-expressing cells in vitro and in vivo conditions (Josimovic-Alasevic et al., 1989). Interestingly, elevated levels of sCD30 have recently been found in the serum of HIV-infected individuals (PiUOlO et al., 1994). Moreover, large numbers of ThP-like CD8+ T cell clones generated from HIV-infected individuals also showed CD30 expression and released detectable amounts of sCD30 in their supernatants (Manetti et al., 1994). These findings prompted us to investigate the possible role of CD30 and CD3OL+ cells in HIV replication. The results showed that cross-linking CD30 with either an agonistic CD30-specific monoclonal antibody (MAb) or fixed CD8+ T cells expressing CD3OL potentiated HIV replication in T cell lines generated from HIV-infected individuals. More importantly, an anti-CDdOL antibody inhibited spontaneous HIV replication in freshly prepared lymph node cells from an HIV-seropositive patient. Thus, CDBOLCD30 interactions may play an important role in enhancing HIV replication, particularly in lymphoid organs, where cell to cell contact can easily occur. Ftesults and Discussion We first searched for the presence of CD30+ cells in the circulation of HIV-infected individuals. Virtually nocirculating CD30+ cells were found in either 16 HIV-seropositive individuals, examined at different stages of HIV infection, or 12 HIV-seronegative controls. However, a variable proportion of CD4+ T cells (ranging from 5%-15%) in both HIV-seroposttive and HIV-seronegative subjects expressed CD30 following in vitro expansion in medium containing phytohemagglutinin (PHA) and interleukin-2 (IL-2). We therefore asked whether triggering of CD30 in naturally infected CD4+ T cell lines from HIV-seropositive individuals could play any role in HIV replication. Polyclonal CD4+ T cell lines, derived from six HIV-infected individuals (four belonging to group II and two to group Ill), and which had been selected for their inability to produce p24 Ag spontaneously in their supernatants, were stimulated with the combination of an insoluble anti-CD3 plus an insoluble anti-CD30 MAb showing agonistic activity. A significant increase of p24 Ag production in comparison with T cells

Immunity

252

Table 1. Synergistic Effect of AntiCD30 Agonistic Antibody on AntiCDSlnduced HIV Replication in T Cells from HIV-Infected Individuals Antibody present in culture

p24 Ag production @g/IO cells)

None Anti-CD3 Anti-CD30 Anti-CD3 + anti-CD30


a)

10.’ I

T cell lineswere generated from HIV-infected individuals by stimulation of PEWS (1 x W/ml) with PHA (0.1% v/v) and IL-2 (20 U/ml). T cell blasts (1 x W/ml) from six lines selected for showing no spontaneous p24 Ag production in their supernatant were cultured IO days later for 3 days in presence of insoluble anti-CD3 MAb, insoluble agonistic anti-CD30 MAb (MM), or both. Cell-free supematants were then assayed for p24 Ag content, as described in Experimental Procedures. Mean values ( f SD) are reported. ‘p
Anti-CD30 with anti-CD3 MAb alone was obsewed (Table 1). In subsequent experiments, polyclonal CD4+ T cell lines were also generated from two HIV-infected individuals belonging to group IV, subgroup C-l. In spite of the fact that these lines showed spontaneous p24 Ag production, stimulation with anti-CD3 plus anti-CD30 MAb again resulted in a significant increase of p24 Ag production in comparison with either unstimulated T cells or T cells stimulated with anti-CWOor anti-CD3 MAb alone. The kinetics of CD30 expression induced by stimulation of T cells from one of these lines with the insoluble anti-CD3 MAb is shown in Figure 1a. CD30 expression became evident on day 1 and peaked on day 2 after activation. Figure 1 b shows the kinetics of p24 Ag production induced in the same line by stimulation with anti-CD3 MAb alone or the combination of anti-CD3 plus anti-CD30 MAb. The potentiating effect of anti-CD30 MAb on p24 Ag production was also maximal on day 2, clearly paralleling the expression of CD30 induced by the anti-CD3 MAb (Figure la). The potentiating effect of anti-CD30 MAb on HIV replication did neither simply reflect an increase in cell proliferation nor a TNFa-mediated stimulation, inasmuch as it was not inhibited by either anti-IL-2 or anti-TNFa antibody (data not shown). In parallel experiments, we have found that CD30 cross-linking induced NF-xB up-regulation in ThO and Th2, but not in Thl , CD4+ T cell clones (P. P. McDonald et al., submitted). This suggests that induction of HIV expression by CD30 triggering may result from binding of NF-KB transcription factors to KB sites present in the HIV long terminal repeat, as it has been shown forthe induction of HIV transcription by TNFa (Duh et al., 1989; Poli et al., 1990; Butera et al., 1993). The next obvious question was to establish whether a similar potentiating effect on HIV replication could be exerted by CD30L-expressing cells. Therefore, we tested freshly prepared peripheral blood mononuclear cells (PBMCs) from normal individuals or patients with chronic lymphocytic leukemia (CLL), tonsil mononuclear cells, as well as a panel of 20 CD4’ and 15 CD8+ T cell clones, for membrane CD3OL expression. Spontaneous CDBOL expression was observed in most B cells from both fresh stimulated

0

1

2

i

Days after activation Figure 1. Kinetics of CD30 Expression Induced by Stimulation with Anti-CD3 MAb and of p24 Ag Production Induced by Costimulation with Agonistic Anti-CD30 MAb Polyclonal Tcell linesweregenerated from an HIV-seropositive individual, as reported in the legend of Table 1. On day 10, cells (1 x WY) were cultured in the absence or presence of insoluble antiCD3 MAb, insoluble antiCD30 MAb, or both. (a) Cytoftuorometrtc pattern of CD30 expression by cells examined before (time 0) and after l-3 days stimulation with insoluble anti-CD3 MN values in parentheses represent numbers of CD4+ T cells expressing CD30. (b) Kinetics of p24 Ag production in parallel unstimulated cultures (closed triangle) or cultures stimulated with anti-CD30 MAb (closed inverted triangle), anti-CD3 MAb (closed circle) or a combination of antiCD3 and anti-CD30 MAb (closed square).

peripheral blood or tonsil suspensions, as well as in established CD8+, but not in CD4+, T cell clones (data not shown). Three CD8+CD30L+ T cell clones, two of which were generated from an HIV-seronegative healthy donor (SR18 and SR92) and one from an HIV-infected individual (lB75), were fixed with paraformaldehyde (0.5%) and cocultured for 48 hr with T cell lines generated from three HIV-seropositive individuals in the presence of insoluble anti-CD3 MAb. Stimulation with anti-CD3 MAb alone induced variable production of p24 Ag, but the addition of fixed CD8+CD30+, but not of CD8+CD30-, clonal T cells

HIV Expression 253

by CD80 Triggering

q Ant&CD301Ab

T

Nom

SR 18

sR92

IB 75

CD30L+

IB54 CDBOL-

CD8+ T-CELLCLONES Figure 2. Potentiating Activity of CD8+ T Cell Clones Expressing CD8OL on HIV Replication in CD4+ T Cell Lines Generated from HIVBeropositive Individuals T cell lines were generated from PBMCs of HIV-infected individuals, as described in the legend of Table 1. T cell blasts (1 e/ml) from each of three CD8+ CD8OL+ and one CDB+CDXrLT cell clones were fixed with 0.5% paraformaldehyde and cocultured for 48 hr with T blasts (W/ml) from each of three different T cell lines in the presence of insofuble anti-CD3 MAb and in the absence or in the presence of a soluble antiCD8OL MAb (M81). Cell-free supernatants were collected and assayed for the content of p24 Ag. Columns represent mean values (f SEM) of p24 Ag concentrations.

Figure 3. Enhancement of HIV Replication byCD8OL B Lymphocytes PBMCs (1 W/ml) from four HIV-infected individuals were stimulated for 6 days with insolubilized anti-CD3 MAb in absence (open bar) or in presenceof CD8OL+ Bcells(lOYml)from aCLLpatient(wide hatching), CD8OL+ plus soluble CD80-lg fusion protein (1 rig/ml) (narrow hatching), CD8OL’ B cells plus antiCD8OL MAb (M81, 1 pa/ml) (reverse hatching), CD8OL- B cells from another CLL patient (closed bar) or recombinant TNFa (100 @ml) (stippled bar). Cell-free supernatants were collected and assayed for type content of p24 Ag. Columns represent mean values ( f SEM)of p24Ag production from the four patients.

promoted consistent and marked increase of p24 Ag production in all three HIV-infected T cell lines, and such a potentiating effect was completely inhibited by the addition of antiCD30L MAb (Figure 2) whereas the addition of an anti-Fas (M3) MAb showing the same isotype had no significant inhibitory effect (data not shown). The increase of p24 Ag production induced by CD30+ cell8 was about 10-l 00 time8 higher than that stimulated by the anti-CD30 MAb, suggesting that CD30 triggering by its natural ligand is more effective than triggering by plate-bound anti-CD30 antibody. Of note also is that one T cell line was generated from the same donor of one of the three CD8+CD30L+ T cell clones (IB), suggesting that CD30-CD30L interaction can promote viral replication in both autologous and allogeneic CD4+T cells. Similar results wereobtained by using PBMC from one patient with CLLshowing CD30L+circulating cells, whereas PBMC from another patient with CLL showing no CD30L+ cells did not induce any increase of HIV replication. Interestingly, in these experiments HIV replication induced by CD30L+ PBMC was significantly higher than that induced in the same system by TNFa. Moreover, HIV replication induced by CD30L-CD30 interaction was inhibited by the addition of either antiCD3OL MAb or a soluble CD30-lg fusion protein (Figure 3). To verify that what occurs in vitro relates to the situation in vivo, we finally assessed the expression of CD30L on the lymph node biopsy specimen from an HIV-seropositive subject. Both the majority of B cells and a proportion of CD8+ cells expressed CD3OL (Figure 4a). The CD8+CD3OL+ cells were CD3+CD16-, a6’ yS- (data not shown), suggesting a T cell nature despite the low expression of CD8

antigen. Interestingly, spontaneous p24 Ag production by lymph node cells from the same patient was remarkably inhibited (about 50%) by addition in culture of antiCD30L MAb, whereas both anti-IL-2 and anti-TNFa MAbs showed poor or no inhibitory effect (Figure 4b). In another series of experiments, freshly derived PBMC from three HIVserofxrsitive subjects, selected for showing spontaneous p24 Ag production, were also incubated with antiCD30L MAb or the soluble CD30-lg fusion protein. Spontaneous p24 Ag production was partially but consistently inhibited by both reagents, whereas neither anti-TNFa MAb nor soluble CD40-lg fusion protein, used as controls, were inhibitory(Table2) suggesting that the interaction between HIVinfected CD4+CD30+ T cells and cells expressing CDBOL can promote HIV replication in vivo, as well. Taken together, the results of this study clearly demonstrate that CD30 triggering induces HIV expression in infected CD4+ T cells. This finding is of particular interest in view of the demonstration that CD30 is preferentially expressed by T cells producing ThP-type cytokines. Indeed, if there is induction of a Th2 bias during HIV infection (Clerici et al., 1993) rapid killing of ThO/Th2 cells at the lymph node level as a consequence of enhanced HIV replication Caused by CD30L-CD30 interaction, likely occurs, thus accounting for the difficulty of proving the validity of the Thl/ThP switch hypothesis (Maggi et al., 1994a; Graziosi et al., 1994). The demonstration that elevated levels of sCD30 can be found in the serum of HIV-infected patients(Pizzolo et al., 1994) is alsoin favorof thispossibility. Inasmuch as the increase of serum sCD30 reflects the bulk of cells expressing CD30 in vivo, even in those

Immunity 254

a)

Table 2. Inhibitory Effect of Anli-CD3OL MAb and of CD30-lg Fusion Pmtein on the Spontaneous p24 Ag Production by T Cells from HIV-Infected Individuals Reagent added in culture None AnliCD3ClL MAb Anti-TNF a MAb

CD3Wg

b)

CD40-IQ

p24 AQ production Experiment 114 52 120

64 105

1

@g/ml) Experiment

2

98 35 90 27 110

Experiment

3

288 139 314 ND ND

PBMC from three HIV-infected individuals, selected for their ability to produce spontaneously p24 Ag in their supernatant, were cultured for 6 days in absence or in presence of antiCD30L MAb (M81; lmmunex Corporation; 1 pg/ml), anti-TNF a MAb (Genzyme Corporation. Cambridge, Massachusetts; 1 rglml), CD30-lg (Immunex Corporation; 1 @ml). or CD40-lg (Immunex Corporation; 1 &ml) fusion protein. Production of p24 Ag was measured in cell-free supernatants, as de scribed in Experimental Procedures. ND, not done.

Nil

Figure 4. lnhibitoly p24 Ag Production Subject

antiCDBOL

an& IL-Z

anliTNF-a

Effect of Anti-CD3OL MAb on the Spontaneous of Lymph Node Cells from an HlV-Sempositive

A biopsy specimen, taken for diagnostic purposes from the cervical lymph nodes of an HlV-seropositive subject, was teased and the resulting cell suspension centrifuged on a Ficoll-Hypaque density gradient. (a) Cell suspensions were examined for the expression ofCD3lIL(MEl) by cytofluorimetric analysis, as described in the legend of Figure 1. (b) Lymph node cell suspensions were cultured in absence or in presence of anti-CD30L (1 pglml), anti-IL-2 (1 @g/ml), or anti-TNFo (1 &ml) MAb, and production of p24 Ag was measured in cell-free supernatants after 5 days.

condition8 in which circulating CD30+ cell are not detectable (Josimovic-Alasevic et al., 1989), the elevated level8 of sCD30 probably result from continuous activation and death of CD4+CD30+ Th2/ThO cells. Such a possibility was recently substantiated by the demonstration that expression of CD30 in CD4+ T cell clones infected with HIV in vitro often preceeds, and associates with, early CD4+ T cell death (E. M. et al., unpublished data). The results of this study also suggest that cells involved in HIV replication induced by CD30 triggering may be either 6 cell8 or CD8’ T cells, inasmuch as both cell types were found to express the CD30L. However, CD9+ T cells usually play a protective role against HIV infection (Brinchmann et al., 1990; Mackewicz and Levy, 1992), at least until they do not switch to a less protective functional phenotype (Maggi et al., 1994b; Manetti et al., 1994). Therefore, B cells and possibly activated macrophages, which have aI80 been shown to express CD3OL mRNA (Smith et al., 1993), are more likely responsible for HIV replication induced in vivo by CD30L-CD30 interaction. Such a mechanism may be particularly relevant in the activation of HIV expression from latently infected CD4+ T cells in lymph nodes where B cell/I-h cell contact continuously occurs. Finally, the demonstration that blocking CD3OL-CD30 interaction can markedly suppress the spontaneous p24 Ag

production in freshly derived lymph node cells from HIVinfected individuals may prompt the consideration of new therapeutic strategies for inhibiting HIV replication. ExPerImental Procedures PHA was purchased from GIBCO Laboratories (Grand Island, New York). Recombinant IL-2 was a gift of Eurocetus (Milano, Italy). AntiCD20, antiCD4, antiCD8, antiCD16, anti+TCR, and anti+TCR MAbs were purchased from Becton-Dickinson (Mountain View, California). Anti-CD3 MAb was purchased from Ortho Pharmaceuticals (Raritan, New Jersey). Anti-IL-2 and anti-TNFa MAbs were purchased by Janssen (Beerse, Belgium) and Genzyme Company (Cambridge, Massachusetts), respectively. Anti-CD30 MAb (Ber-H2) was purchased by DAK0 (Glostrup, Denmark). Anti-CD30 MAb with agonistic activity (M44), anti-Fas (M3), antiCD3OL MAbs(MBOand MEl), and the CD3& Ig and the CD40-lg fusion protein were provided by lmmunex Corporation (Seattle, Washington). Subjects PBMCs were obtained from 27 HIV-infected patients, 12 HIVseronegative healthy volunteers, and 3 patients with CLL. HIV-infected individuals (5) were classified in the group II. 5 in the group Ill, and 17 in the group IV, according to the Centers for Disease Control (CDC) criteria (Centers for Disease Controls and Prevention, lg87). Group IV patients (4) belonged to subgroup A, 10 to subgroup C-l, and 3 to subgroup D. A biopsy specimen was taken for diagnostic purposes from the cervical lymph nodes of a HIV-seropositive subject belonging to group Ill. Histological examination revealed a pattern of nonspecific adenitis with strong follicular hypsrplasia, suggesting classical HIVrelated lymphoadenopathy. Mononuclear cells from palatin tonsils of a child who underwent tonsillectomy, as well as mononuclear cells from three patients with CLL were also used throughout the study. All patients and controls gave informed consent.

Cell

Phenotyplng CD30 expression by T cells of HIV-infected patients and controls was performed on a Cytomn Absolute cytofluorimeter (Orlho Pharmaceuticals, Raritan, New Jersey) by using fluoresceinated antieD MAb and phycoerylhrinated anti-CD4 or anti-CD8 MAb. CD30L expression was evaluated by using an anli-CD30L MAb (M81, IgG2b). followed by fluoresceinated anti-mouse lg2b goat antibody (Southern Biotechnology Associates; Birmingham, Alabama) and phycoetylhrinated anti-CD20 (Leu 16, IgGl), anti-CD4 (Leu 3a, IgGl), or anti-CD8 (Leu 2a, IgG1) MAbs.

Generation of 1 Cell Lines snd T Cell Clonem T cell lines were generated

from 6 HIV-infected

individuals

by stimula-

HIV Expression 255

by CD30 Triggering

tion of PBMC (1 x Iv/ml) with PHA (0.1% v/v) and recombinant IL-2 (20 U/ml). T cell clones were generated from PBMC of one HIVseropositive and one HIV-seronegative by a high efficiency cloning technique (Macchia et al., 1993). In brief, PBMCs were seeded under limiting dilution conditions (0.3 cell/well) in round-bottomed microwells containing 105 irradiated (6.000 rads) allogeneic spleen cells (as feeder cells) and PHA (1% v/v) in a final volume of 0.2 ml RPM1 1640 medium supplemented with 2 mM Lglutamine, 2 x IO* M 2-ME (complete medium) containing human recombinant IL-2 (20 U/ml), and 10% fetal calf serum (HyClone Laboratories, Logan, Utah). Growing microcultures were then supplemented at weekfy intervals with IL-2 (20 U/ml) and IO6 irradiated feeder cells. Phenotyping of T cell clones was performed by using fluorescein-conjugated anti-CD4 and phycoerythrinated anti-CD6 MAbs. induction of p24 Ag Productlon by T Cell Lines from HIV-Infected tndlvlduals T cell blasts (10S/ml) from polyclonal T cell lines generated from HIVinfected individuals were cultured for 3 days in presence of insoluble anti-CD3 MAb, insoluble anti-CD30 MAb showing agonistic activity (M44), or both. T cell blasts (iv/ml) from the same T cell lines were also cultured for 2 days with T cell blasts (1 p/ml) from three paraformaldehyde-fixed (0.5%) CD6* CD3OL+ T cell clones in presence or in absence of insoluble anti-CD3 MAb. Cell-free supernatants were collected and assayed for the content of p24 Ag by an appropriate ELISA (HIVAG-1 Monoclonal, Abbott; Wiesabaden-Delkenheim, Federal Republic of Germany). Acknowledgments This work was supported by grants from Associazione ltaliana Ricerca Cancro, Minister0 della Sanita (Progetto AIDS), and European Community Project (Biotechnology: “Immunotoxicology in vitro?. Received

March

30, 1995; revised

June 21, 1995.

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